Rac1 selective activation promotes axonal regeneration after optic nerve crush in Brainbow mice.

<p>In Brainbow mice the injection of the AAV-Cre-GFP around the day of crush triggers a genetic recombination that leads to YFP expression (white false color) only in surviving neurons. We injected either the Tat-Rac1 mutants, WT or vehicle on the day of crush (day 0) and on day 2 and studied regeneration 15 days post lesion. A scheme of the treatment is in A. Nerves were acquired and studied by confocal microscopy. Single examples of whole mounted nerves after mosaic merge reconstruction are given in B to E, and are relative to vehicle (B), Rac1WT (C), L61F37A mutant (D) and L61Y40C mutant (E) double injections. The crush sites of B, C, D and E are enlarged in F, G, H and I respectively. By comparison of the panels it is clear that after treatment with L61F37A a higher number of axons is able to cross the crush site and run distally (D and H). Scale bars 100 µm.</p

Rac1 selective activation prevents the dendrite atrophy occurring after crush.

<p>Representative confocal maximum projections of YFPH mouse RGCs 15 days after crush and double injection of either vehicle or CA or DN mutants. A normal RGC is shown for comparison (A). After Rac1L61F37A and Y40C treatment (D and E respectively) the dendritic atrophy is prevented, whereas the same extent of degeneration was found in control (B) and after DN treatment (C). The plots relative to the Sholl analysis for the different treatments are shown in F–G: (F) the maximum number of intersections, the ramification index and the critical value were used for statistical evaluation (10 to 20 neurons per treatment), whereas (G) the N of intersections against the distance from the soma shows the morphological changes along the whole dendritic tree. Scale bar 20 µm. # p<0,01 versus the control, the L61F37A and the L61Y40C; *p<0,05 versus the indicated group (one way ANOVA followed by LSD post hoc test).</p

Quantification of axonal regeneration after optic nerve crush and Rac1 selective activation in Brainbow mice.

<p>Brainbow mouse optic nerves were crushed at day 0 and, after one or more injections of either Rac1 mutants, WT or vehicle, were dissected at 15 or 30 days post crush in order to investigate regeneration. A scheme of the different treatments is given in A. Nerves were studied by confocal microscopy and the results of the regeneration study are plotted in B, C and D. Only the double injection of L61F37A was able to increase the average number of axons crossing the crush site per 100 µm of nerve z-section (B) at 15 days post lesion. The number of regenerating axons is higher than control also after 2 and 5 injections of L61F37A at 30 dpl. The data at 15 days are confirmed also by the analysis of length distribution in the entire distal stump (C). The same analysis at 30 days (D) revealed that, despite after 2 and 5 L61F37A injections the total number of regenerating neurons are similar, the repetitive treatment resulted in longer axons. Since we found no differences between the various vehicle injection protocols of treatment at 15 and 30 days, we put together the data of the controls on the same column/curve (n = 6 and 8). Data are mean ±SEM. N is in brackets. ^#p<0.01, *p<0.05 by ANOVA (LSD post hoc test).</p

Phosphorylation of Pak1 and upregulation of ERK1/2 after injections of Rac1 mutants.

<p>Retina sections were immunostained by antibodies against the pan-specific and the phosphorylated form of Pak1 (T212), ERK1/2 and JNK. Retinas were dissected and immunostained 3 days after optic nerve crush and ivit treatment with either vehicle, or Rac1WT, or L61F37A or L61Y40C. Some representative relevant images are shown in A to J, N and O. (A–B) Colocalization of phospho-Pak1 (Pak1-p, red) and βIII tubulin (green) indicates that the L61F37A resulted in Pak1-p increase in RGCs, confirmed also by the lack of colocalization with GFAP (C–D). (E–F) Colocalization of phospho-ERK (ERK1/2-p, red) and GFAP (green) indicates that the L61Y40C mutant activated ERK1/2 in retinal glial cells. (G–H) The lack of colocalization between MAP2 (blue) and ERK1/2-p after Y40C treatment indicates that this protein is not activated in neurons. A control is also shown (I–J) where ERK1/2-p positivity is very low. (N–O) Histag staining after L61Y40C treatment showed a clear positivity in RGCs, meanwhile the ERK1/2-p positivity pattern was still glial-like. (K) Semi-quantitative expression of total and phosphorylated level of Pak1, ERK1/2, and JNK measured in correspondence of the ganglion cell layer and normalized on the signal of the normal eye (n = 9 to 15 confocal stacks from 3 to 6 animals). (L) Ratio between the normalized intensity of phosphorylated and total forms of Pak1, ERK1/2 and JNK gives an indication about the degree of kinase activity. Scale bar 30 µm. GCL: ganglion cell layer. (M) We hypothesize that L61F37A effect on survival might be related to the increased expression of ERK1/2 and increased Pak1 phosphorylation in neurons. Meanwhile, L61Y40C is most likely boosting survival through the activation of ERK1/2 in astrocytes.</p

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