Additional file 1: of Rac1 activation links tau hyperphosphorylation and Aβ dysmetabolism in Alzheimer’s disease

Figure S1. Rac1 mutant peptides have high penetration due to the TAT sequence. (A-C) Representative confocal images of cortical neurons treated at DIV3 with different concentrations of TAT-GFP: 5 μM (A), 10 μM (B, C). After treatment, cells were fixed and stained for visualization of dendrites (MAP2) and nuclei (DAPI). Confocal analysis showed that TAT-GFP was internalized (single plane), also in live cells directly imaged 1h after treatment. Scale bars 10 μm. (D) MTT assay on primary cortical neurons after 24h from the administration of 2 μM Rac1 mutant peptides. The cell viability is expressed as % as compared to control. The data represented are mean ±SEM of four independent experiments, each done in triplicate. Figure S2. Aβ1-42 administration does not interfere with Rac1 localization or activation. (A) MTT assay on primary cortical neurons after 24h Aβ1-42 treatment at the indicated concentrations The Aβ peptide suspension was incubated 12h at 4°C prior treatment. The cell viability is expressed as % as compared to control. The data represented are mean ±SEM of four independent experiments, each done in triplicate. One-sample t test to a hypothetical mean of 100% was performed. (B) Representative dot-blot analysis of Aβ1-42 preparations with 6E10 and A11 antibodies. The protein concentration was 0.12 μg for 6E10 and 0.72 μg for A11 (C) Representative confocal images of primary cortical neurons treated with 0.1 μM Aβ1-42 between DIV11 and DIV14. Cells were stained against Rac1-GTP, F-actin, and neurofilament. Scale bars 30 μm. Figure S3. Efficacy of the subcellular fractionation. Representative blots of the subcellular fractionation experiments showing the levels of GluR1, LaminB, and SET in the membrane and nuclear fractions of SH-SY5Y cells. Four independent samples were assessed for the 2 fractions. Figure S4. Tau induced hyperphosphorylation does not alter Rac1 levels or activation. (A) Representative confocal pictures of mature cortical neurons treated with 10nM OA for 6h and immunostained against pS262 tau. Scale bar 30 μm. (B-C) Tau pS262 phosphorylation was analysed by western blot after 3 and 6h from OA administration. The data represented are mean with SEM of four or six independent experiments (3h treatment n=6, 6h treatment n=4). (D-E) Rac1-GTP pull done assay was performed after 3 and 6h from OA administration. The data represented are mean with SEM of three independent experiments. ns, not significant. Asterisks indicate unspecific bands. (DOCX 3215 kb

PrP immunohistochemistry of muscle samples from Alpine brown cow experimentally infected with BASE (#995).

<p>PrP immunohistochemistry of muscle samples from Alpine brown cow experimentally infected with BASE (#995).</p

PrP immunohistochemistry of muscle samples from the Holstein-Friesian cow affected by natural BASE (#126752/09).

<p>PrP immunohistochemistry of muscle samples from the Holstein-Friesian cow affected by natural BASE (#126752/09).</p

Transmission of BASE to Tgbov XV mice following inoculation of muscles from experimentally and naturally affected cattle.

<p>(A) Lesion profiles of mice infected with brain tissue from natural BASE (blue line) and BSE (green line), <i>longissimus dorsi</i> muscle from experimental BASE (red line) and <i>intercostalis</i> (bordeaux line) and <i>gluteus</i> (yellow line) muscles from natural BASE. Vacuolation was scored on a scale of 0–5 in the following brain areas: 1, dorsal medulla; 2, cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7, septum; 8, retrosplenial and adjacent motor cortex; and 9, cingulated and adjacent motor cortex. Data are mean ± s.e.m. (B) Western blot analysis of proteinase K-digested brain samples of mice infected with brain homogenates from cattle with natural BASE (#12966/07) and BSE, <i>longissimus dorsi</i> muscle from cattle with experimental BASE (#995) and two different muscles from natural BASE (#12699/07). (C–M) Neuropathological changes of mice infected with brain (C–E) and muscle from cattle with experimental BASE (F–H), and muscle from cattle with natural BASE (I–M). Micrographs were obtained from corresponding areas of the thalamic region stained with haematoxylin-eosin (C,F,I) or labeled with the anti-PrP antibody 6H4. The severity of vacuolation and the type of PrP deposition, characterized by diffuse immunostaining of the neuropil with focal enhancement, is similar in all the samples analyzed. Scale bar = 100 µm.</p

PrP deposition in the muscles of natural BASE cattle.

<p>(A–C) PrP deposits in <i>peroneus</i> muscle from a cattle with natural BASE (#126752/09). The PrP-imunoreactive material was found in isolated muscle fibers with a scattered distribution (A,C) and was localized inside the cytoplasm in the form of small amorphous aggregates or granular deposits (B). Scale bar = 100 µm for figure A and C;  = 20 µm for figure B.</p

Survival Curves and PrP^Sc Types for Inbred Mice Following Primary Transmission of BSE and BASE

<div><p>(A–D) Survival curves are for (A) SJL mice, (B) C57Bl/6 mice, (C) RIII mice, and (D) VM mice.</p><p>(E) Western blot analysis of proteinase K-treated brain homogenates from SJL, RIII, C57Bl/6, and VM mice challenged with BSE.</p><p>(F) Western blot analysis of brain homogenates from SJL, RIII, C57Bl/6, and VM mice challenged with BASE and a BSE-infected SJL mouse used as positive control (last lane). The first two lanes correspond to the same SJL mouse prior to and after proteinase K digestion, while all other samples were digested with proteinase K. The blots were probed with the anti-PrP antibody 6H4.</p></div

Primary Transmission of BSE and BASE to Tgbov XV Mice

<div><p>(A) Survival curves and (B) lesion profiles for mice infected with BSE and BASE. (B) Vacuolation was scored on a scale of 0–5 in the following brain areas: 1, dorsal medulla; 2, cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7 septum; 8, retrosplenial and adjacent motor cortex; and 9, cingulate and adjacent motor cortex. Data are mean ± s.e.m.</p><p>(C–J) Neuropathological changes in mice infected with BSE (C–F) and BASE (G–J). Micrographs were obtained from corresponding areas of the hippocampal region and cerebral cortex stained with haematoxylin-eosin (C, D, G, and H), or labeled with the anti-PrP antibody 6H4 (E, F, I, and J). The neuropathological profile of BSE-infected mice is marked by the presence of numerous amyloid plaques while spongiform changes are mild (C and D). Conversely, mice challenged with BASE show a severe vacuolation in the absence of amyloid deposits (G and H). PrP immunohistochemistry shows uni- and multicentric amyloid plaques associated with granular deposits in BSE-infected mice (E and F) and diffuse immunostaining of the neuropil with focal enhancement in mice challenged with BASE (I and J). Scale bar: 200 μm (C and G); 50 μm (D and H); 500 μm (E and I); 100 μm (F and J).</p><p>(K and L) Western blot analysis of proteinase K-treated brain homogenates from (K) Tgbov XV mice challenged with BSE and BASE, prior (left panel) and after (right panel) deglycosylation with PNGase; and (L) cattle with BSE and BASE. The blots were probed with the anti-PrP antibody 6H4. The samples in (L) correspond to the actual inocula used for the transmission studies.</p></div

Lesion Profiles for Inbred Mice Following Primary Transmission of BSE

<p>(A–D) Lesion profiles are for (A) SJL mice, (B) C57Bl/6 mice, (C) RIII mice, and (D) VM mice. The lesion profile was determined as specified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030031#ppat-0030031-g001" target="_blank">Figure 1</a>. Data are mean ± s.e.m.</p

Brain MRI of Tgbov XV Mice Infected with BSE and BASE

<p>T2-weighted images of anterior-to-posterior coronal planes of (A–C) BASE-infected mouse; (D–F) BSE-infected mouse; (G–I) uninfected Tgbov XV mouse. Both BSE- and BASE-infected mice show high signal intensity in the septal region: arrowheads in (A) and (D); and cerebellum: arrowheads in (C) and (F) compared to control (G) and (I). In addition, mice challenged with BASE exhibit scattered hyperintense areas in frontal regions: arrows in (A); and midbrain: arrows in (B) that are absent in BSE-infected and uninfected mice.</p