Expression of human tau in the optic nerve of mice transgenic for human mutant P301S tau.

<p>(A–C), Staining for human tau (A, green) and βIII tubulin (B, red) showed co-localisation in the axons of the optic nerve (C, overlay image of A and B; example from 1 month old mouse). (D–F), Staining for tau phosphorylated at S202/T205 (D, green) and βIII tubulin (E, red) showed co-localisation in the axons of the optic nerve (F, overlay image of D and E; example from 5 month old mouse). Arrows indicate examples of co-localisation. Scale bar, 20 µm.</p

Quantification Methodology.

<p>(A), Retinal ganglion cell (RGC) survival following excitotoxic injury <i>in vivo</i> was quantified using retinal flat mounts immunohistochemically labelled for NeuN. Twelve images (3 per quadrant at central, medial and peripheral locations; approximate positions defined by boxes) were captured per retina using a 40× objective; NeuN-positive nuclei were counted in each image and their average number calculated for each retina. RGC loss was calculated compared to NeuN counts from the uninjured contralateral eye. (B), Axonal transport of fluorescent cholera toxin B (CTB) in the optic nerve was quantified by measuring average fluorescence intensity across the width of the optic nerve at 100 µm intervals along the full length of each nerve. A representative image is shown, with the white lines indicating example regions where average fluorescence intensity was measured. Scale bar, 100 µm.</p

Anterograde axonal transport is reduced in optic nerve of mice transgenic for human mutant P301S tau mice.

<p>Fluorescent cholera toxin B was injected unilaterally into the vitreous and the amount transported in an anterograde direction measured in 5-month-old (A), 3-month-old (C) and 1-month-old (E) P301S tau transgenic and C57/Bl6 control mice. Fluorescence intensity appeared lower in transgenic mice at all ages, compared to controls. In optic nerves not exposed to cholera toxin B (negative control), only background fluorescence was measured (A,C,E). Statistical analysis of the area under the fluorescence intensity curve for each individual showed a significant reduction of anterograde axonal transport in optic nerves from P301S tau transgenic mice at 5 months (B) and at 3 months (D), but not at 1 month (F). Data are presented as mean±SEM.</p

Binding of cholera toxin B (CTB) protein to GM1 receptor in P301S tissue.

<p>Fixed retinal (A–B) and brain (C–F) tissue was exposed to fluorescently tagged CTB (red) in order to visualise GM1 receptor binding in both C57/Bl6 control tissue (A, C–D) and P301S tissue (B, E–F). CTB binding was observed in RGCs within the retina (A–B), counterstained for the marker βIII tubulin (green; arrows indicate co-localisation), and punctate staining within the inner retina was also seen. Nuclei were counterstained with DAPI (blue). No difference in the pattern of CTB binding to the GM1 receptor in the retina was found between control (A) and P301S (B) tissue. Furthermore, no difference in the pattern of CTB binding to the GM1 receptor in the superior colliculus (outlined) of the brain was observed between control (D) and P301S (F) tissue. The RGC axon terminals in the superior colliculus were counterstained for the marker vGluT2 (vesicular glutamate transporter 2; green) in both control (C) and P301S (E) tissue. Scale bar, 25 µm A–B, 500 µm C–F.</p

Retrograde axonal transport is reduced in optic nerve of mice transgenic for human mutant P301S tau.

<p>Fluorescent cholera toxin B was injected bilaterally into the superior colliculus and the amount transported measured in 5-month-old (A) and 3-month-old (C) P301S tau transgenic and C57/Bl6 control mice. Fluorescence intensity was lower along the length of the optic nerve in transgenic mice at all ages, compared to C57/Bl6 controls. Statistical analysis of the area under the fluorescence intensity curve for each individual showed a significant reduction in retrograde axonal transport in optic nerves from P301S tau transgenic mice at 5 months (B) and at 3 months (D), compared to controls. Data are presented as mean±SEM.</p

Reduced axonal transport in optic nerve of mice transgenic for human mutant P301S tau increases neuronal susceptibility to injury.

<p>Retinal ganglion cell (RGC) survival was quantified following a mild unilateral excitotoxic injury of the left eye (LE) by counting NeuN-positive nuclei in the RGC layer of the whole-mounted retina of 1, 3 and 5 month old mice (A–D). Percentage RGC loss was calculated by comparing the number of surviving RGCs in injured retinas to that of the uninjured right eye (RE). Representative images of NeuN-positive nuclei in injured P301S tau transgenic (A) and C57/Bl6 (C) retinas from 5 month old animals, compared to uninjured contralateral retinas (B and D), are shown as an example. Statistical analysis revealed a significant increase in RGC death following mild excitotoxic injury in P301S tau transgenic retinas, compared to C57/Bl6 control retinas (E), at both 3 months and 5 months of age; however no difference in RGC excitotoxic death was found at 1 month of age between control and transgenic retinas. N-methyl-D-aspartic acid (NMDA) was used as the excitotoxin. Scale bar, 100 µm.</p

Antibodies used in the present study indicating source, manufacturer, catalogue number and dilution.

<p>Antibodies used in the present study indicating source, manufacturer, catalogue number and dilution.</p

Integrin distribution within the retina.

<p>The images show the integrin label in green, βIII-Tubulin in red and DAPI staining in blue. Distribution of αV, α1, α3, α5 in A, B,C,D (scale bar in D for all α) and labelling for β1 and β3 in E, F (scale bar in F for both β). Arrowheads point to the retinal pigment epithelium. The α integrins and β1 were mainly located in the retinal ganglion cell layer, endothelium of the vessels located closed to the RGCs wile β3 was found mainly within the inner plexiform layer. The retina layers are indicated in E. Outer segments (OS), outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL).</p

Influence of the substrata and cell distribution.

<p>In all cases the statistical difference was (p<0.05). <b>A: Influence of the substrata on cell survival.</b> The influence of the substrate on the number of observed cells PL and L (•) show significant differences compared to the other substrates; CI () shows significant differences in the number of cells compared to the other substrates and CIV and F (#) have significant differences compared to the other substrates B: Distribution of RGC complexity (number of neurites per cell). B1: Low level of complexity. Significant differences were found for PL and L (•) against the rest of the substrates; CI () against the rest of substrates and CIV and F (#) with the rest of the substrates. <b>B2: Medium level of complexity.</b> Significant differences were found for each substrate compared to the other substrates. B3: High level of complexity. Significant differences were found for PL, L and CIV (•) against CI and F; CI () and F (#) was significantly different to all other substrates. C: Distribution of RGC neurites length between the different substrates. C1: Short length RGCs growing in PL and L (•) are significantly different to the other substrates; and the rest of the RGCs growing in CI (), CIV (#), or F (##) each was different to the other substrates. <b>C2 Medium length.</b> CI ($) was significantly different to the other substrates, but there was no difference between them PL, L, CIV and F (•). <b>C3 Long length</b> neurites were more frequent when RGCs were plated on L (••) and this difference was significant compared to the other substrates. PL and F (•) were the second substrate in which long neurites were present; CI and CIV had significantly fewer long neurites.</p

Distribution of β integrins and pY397 FAK in RGCs growing in laminin.

<p>Paired images A-B, C-D, and E-F of RGCs labelled with a βIII-tubulin antibody in red (A,C,E), and β1 (B) and β3 (D) integrins and pY397 FAK (F) in green. Note that the long RGC neurites pointed out with arrows in C and E are not labelled with either β3 or pY397 FAK while the rest of RGCs and their neurites expressed the integrins and were labelled for phosphorylated FAK. Scale bar for all pictures in F.</p