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

Glutamatergic synaptic components in cell membranes of cocultured myotubes.

<p>A: Membrane proteins co-immunoprecipitated by anti-GluR1 antibody were analyzed by immunoblotting using rapsyn antibody. Results show that GluR1 strongly interacted with rapsyn only in cocultured myotubes. Western blot analysis of beta III-tubulin confirmed the presence of neuronal cells only in coculture cell extracts. B: Membrane proteins co-immunoprecipitated by anti-rapsyn antibody were analyzed by immunoblotting using antibodies against GluR1, SAP97, stargazin and PSD95. Results show that both cocoltured myotubes and pure myotubes expressed PSD95, stargazin and SAP97 at the cell membrane. Conversely GluR1 was strongly expressed only in cocultured myotubes. Immunoreactivity of rapsyn-interacting stargazin increased in cocoltured myotubes while that of SAP97 decreased. No change was observed in PSD95 immunoreactivity. C: Confocal images showing the distribution of GluR1, rapsyn, SAP97, stargazin and PSD95 in myotubes cocultured with neurons for 7 days and immunostained. Rapsyn, stargazin and PSD95 colocalize with the receptors, while SAP97 shows a diffuse distribution.</p

Glutamatergic Neurons Induce Expression of Functional Glutamatergic Synapses in Primary Myotubes

<div><h3>Background</h3><p>The functioning of the nervous system depends upon the specificity of its synaptic contacts. The mechanisms triggering the expression of the appropriate receptors on postsynaptic membrane and the role of the presynaptic partner in the differentiation of postsynaptic structures are little known.</p> <h3>Methods and Findings</h3><p>To address these questions we cocultured murine primary muscle cells with several glutamatergic neurons, either cortical, cerebellar or hippocampal. Immunofluorescence and electrophysiology analyses revealed that functional excitatory synaptic contacts were formed between glutamatergic neurons and muscle cells. Moreover, immunoprecipitation and immunofluorescence experiments showed that typical anchoring proteins of central excitatory synapses coimmunoprecipitate and colocalize with rapsyn, the acetylcholine receptor anchoring protein at the neuromuscular junction.</p> <h3>Conclusions</h3><p>These results support an important role of the presynaptic partner in the induction and differentiation of the postsynaptic structures.</p> </div

Time course of synaptic contacts formation in cocultures.

<p>Examples of cortical neurons cocultured with myotubes for 3 (A–D) and 8 days (E–L), fixed, immunostained and studied by confocal microscopy. AMPARs (GluR1 subunit) are in green, axonal neurofilaments and terminations are in red (NF, SV2). At 3 days AMPARs are diffusely distributed (A, C) and myotubes often receive multiple synaptic contacts (B, D). At 8 days AMPARs form clusters (E, I) near or under the terminations (G, K). The Vesicular Glutamate transporter 2 (VGluT2, blue) confirm that the synaptic contact is glutamatergic (H, L). Scale bars 10 µm.</p

Primary myotubes cultured without neurons do not express AMPARs.

<p>Primary myotubes were cultured in absence of neuron and after differentiation, they were treated with the neuronal medium for 1 (A–C), 3 (D–F) and 5 (G–I) days. After fixation and staining for AMPARs (GluR1 subunit, central panels) and AChRs (α-bungarotoxin, left side panels), plates were acquired by confocal microscopy. Right side panels are the overlay of left side and central panels. AchRs are diffusely and widely distributed on muscle surface (A, D, G), whereas no GluR1 positivity was found in all the days examined (B, E, H). Scale bars 10 µm.</p

Cortical neurons form fully functional glutamatergic synapses with myotubes.

<p>In A a scheme of the coculture plate shows how the stimulus was applied to axons crossing the teflon barrier. Calcium-dependent fluorescence variations (B) and myotube shortening during contraction (C) have been measured during electrical stimulation while myotubes were sequentially bathed in saline, treated with Curare, treated with AMPAR antagonist, and after washout. In B an example of myotube fluorescence is also shown for each condition. In all the experiments the treatment with the AMPAR antagonist GYKI 52466 gave a complete block of calcium transients and myotube shortening, demonstrating that synapses are functional and glutamatergic. N = 3, *p<0,05, #p<0,01 by t-test (paired sample, 2 tailed).</p

Glutamatergic neurons from different brain areas form synaptic contacts with myotubes in vitro.

<p>Confocal images showing synapses between myotubes and glutamatergic neurons from cortex (A–F), cerebellum (G–L) or hippocampus (M–O) after 9 days of coculture and immunostained. AMPARs (GluR1 subunit) are in green, axonal neurofilaments and terminations are in red (NF, SV2), whereas AChRs are in cyan (α-bungarotoxin). AChRs are diffusely distributed on myotube surface, whereas AMPARs form clusters that are often near to the axonal termination. Scale bars 10 µm.</p