The Sg4E-BP variant mimicking hyperphosphorylation inhibits protein synthesis in sea urchin cell-free cap-dependent translation system.

<p>Different amounts (20; 100; 200 or 1,000 ng) of recombinant proteins (GST, GST-Sg4E-BP WT (WT), GST-Sg4E-BP YALA (YALA), GST-Sg4E-BP 4xA (4xA), GST-Sg4E-BP 4xE (4xE)) were added to the fertilized cell-free translation system and Luciferase activity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005070#s4" target="_blank">Materials and Methods</a> after addition of the Cap+mRNA encoding Renilla Luciferase. The Luciferase activity is represented in RLU (Relative Light Units). Error bars represent the standard deviation (s.d.) of duplicates.</p

Translation inhibition is rescued by GST-mIF4E.

<p>200 ng of GST recombinant proteins (GST, GST-Sg4E-BP WT (WT), GST-Sg4E-BP YALA (YALA), GST-Sg4E-BP 4xA (4xA), GST-Sg4E-BP 4xE (4xE)) were added to the fertilized cell-free translation system with (lanes 8–12) or without (lanes 2–6) a 5 min pre-incubation with 500 ng of GST-mIF4E (eIF4E) and Luciferase activity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005070#s4" target="_blank">Materials and Methods</a> after addition of a cap mRNA encoding Renilla Luciferase. The Luciferase activity is represented in RLU (Relative Light Units). Error bars represent the standard deviation (s.d.) of duplicates.</p

The variant mimicking hyperphosphorylation of Sg4E-BP also binds to eIF4E.

<p>(A) The S/T-E Sg4E-BP mutated at all four phosphorylation sites (4xE) binds to eIF4E. GST-mIF4E (eIF4E) was incubated with m⁷GTP sepharose beads (lanes 1–5) and the GST recombinant proteins were added: GST, GST-Sg4E-BP WT (WT), GST-Sg4E-BP YALA (YALA), GST-Sg4E-BP 4xA (4xA), GST-Sg4E-BP 4xE (4xE). Complexes were affinity purified and analysed by Western blot using a GST antibody. Lanes 6–10, GST-mIF4E was omitted as control for binding specificity. Inputs are shown on the right panel (lanes 11–16). They represent 10% of the volume used in the experiment. (B) Binding between the GST recombinant proteins and GST-mIF4E was analysed by quantification of the signals obtained on the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005070#pone-0005070-g006" target="_blank">Fig 6.A</a>, using Image Quant software. Error bars represent the standard deviation (s.d.) of two experiments. Significance was assessed using Fisher's <i>F</i>-test and Student's <i>t</i>-test. ^*<i>P</i><0.01 significant difference between GST or GST-Sg4E-BP YALA with GST-Sg4E-BP WT.</p

Wild type Sg4E-BP and the S/T-E variant inhibit eIF4E/eIF4G association with the same efficiency.

<p>Wild type (A) and the 4xE variant of Sg4E-BP (B) inhibit eIF4E/eIF4G association. The m⁷GTP sepharose beads was incubated (lanes 1–7) or not (lanes 8–9) with GST-mIF4E (eIF4E). Then, recombinant proteins GST-Sg4E-BP WT (WT, A) or GST-Sg4E-BP WT 4xE (4xE, B) and GST-SgIF4G (eIF4G) were added separately for lanes 1–2 and lanes 8–9, and together for other lanes (3-4-5-6-7). We used the same amount of GST-Sg4E-BP and GST-SgIF4G in lane 5, a 2-fold ratio in lanes 4 and 6 and a 10-fold ratio in lanes 3 and 7. Proteins were affinity-purified using m⁷GTP sepharose beads and were analysed by immunoblotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005070#s4" target="_blank">Materials and Methods</a> using an anti-GST antibody. Affinity-purified proteins were compared with the GST-fusion proteins loaded separately (lanes 10–12). Inputs represent 10% of the volume used in the experiment.</p

Recombinant GST-Sg4E-BP (4E-BP) interacts with endogenous SgIF4E isoforms in sea urchin extracts.

<p>After incubation of the GST alone (lanes 1–2) or the GST-Sg4E-BP protein (lanes 3–4) in extract prepared from unfertilized eggs (UF, lanes 1 and 3) or from 60 minutes post-fertilization embryos (F, lanes 2 and 4), proteins were affinity purified using Gluthatione Sepharose 4B beads, resolved by 15% SDS-PAGE, analysed by immunoblotting and detected by chemiluminescence using an anti-GST antibody (top and intermediate panels) or anti-eIF4E antibody (bottom panels) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005070#s4" target="_blank">Materials and Methods</a>. SgIF4E that co-purified with GST-Sg4E-BP (lanes 3 and 4) was compared with the endogenous SgIF4E detected in 10 µg of total protein extracts (corresponding to 0,5% of the volume used for the purification) loaded separately (lanes 5–6).</p

Amino acid sequences of 4E-BP proteins.

<p>Sg4E-BP protein was predicted from the cDNA and aligned with the three human 4E-BPs, <i>Ciona intestinalis</i> 4E-BP, <i>Drosophila melanogaster</i> Thor, <i>Aplysia californica</i> 4E-BP and <i>Nematostella vectensis</i> 4E-BP. Accession numbers of human proteins are: 4E-BP1 (NP_004086), 4E-BP2 (Q13542), 4E-BP3 (NP_003723) on NCBI. The Accession number is NP_477295 for <i>Drosophila melanogaster</i> (NCBI), 299279 for <i>Ciona intestinalis</i> (<a href="http://genome.jgi-psf.org/Cioin2/" target="_blank">http://genome.jgi-psf.org/Cioin2/</a>) and SB_47700 for <i>Nematostella vectensis</i> (<a href="http://www.stellabase.org/" target="_blank">http://www.stellabase.org/</a>). The <i>Aplysia californica</i> sequence 4E-BP was obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005070#pone.0005070-Carroll1" target="_blank">[44]</a>. The four residues known to be phosphorylated on human 4E-BPs and conserved on sea urchin are indicated by stars. Identical and conserved amino acid residues are on black and grey background, respectively. The common eIF4E motif indicated by YxxxxLΦ (where x is any amino acid and Φ is an hydrophobic residue), the RAIP motif and the TOS (TOR signalling) site are denoted with a line above.</p

Microgliosis in end-stage haSN(A53T) transgenic mouse brain is unaltered by high LRRK2 levels.

<p>DAB-immunohistochemistry for Iba1 shows activated microglia on a representative sagittal brain section of a haSN(A53T) mouse (A and 20×higher magnification from brainstem in B) and a haSN(A53T)/hLRRK2(G2019S) double transgenic mouse (C and 20×higher magnification from brainstem in D). (E) Quantification of the brainstem results. Values represent % of the area in the brainstem that is covered by Iba1-positive microglia. p-value (p = 0.179) was determined by two-tailed, unequal variances Student’s t-test. Dots represent quantifications of single individuals. Control images obtained from a separate experiment but from littermate hLRRK2(G2019S) single transgenic (F and 20×higher magnification from brainstem in G) and from non-transgenic wildtype littermate control (Ntg) (H and 20×higher magnification from brainstem in I) mouse.</p

High LRRK2 transgene levels do not exacerbate α-synuclein-driven phenotypes.

<p>(A) Schematic representation of the four different transgenic lines used to generate double transgenics. (B) 3-Step accelerated rotarod performance of females and males comparing single and double transgenics. The different genotypes and the number of mice per genotype are indicated. p-values were determined by repeated measures ANOVA (group effects for the respective panels: 1: F(1,22) = 0.483, p = 0.494; 2: F(1,26) = 0.000, p = 0.983; 3: F(1,11) = 0.738, p = 0.409; 4: F(1,22) = 2.048, p = 0.166; 5: F(1,16) = 1.255, p = 0.279; 6: F(1,27) = 5.171, p = 0.031). (C) Kaplan-Meier curves showing the time-of-sacrifice when mice had to be killed because of too severe motor deficits (1 = 100% and 0 = 0% of mice alive). The different genotypes, gender, number of mice per genotype and the p-values (nonparametric Kaplan-Meier) are indicated.</p

aSN and phospho-S129-aSN protein levels in spinal cord and forebrain of end-stage disease single and double transgenic mice.

<p>Tris-soluble and -insoluble fractions of spinal cord and forebrain lysates were immunoblotted and stained with antibodies detecting total α-synuclein (aSN) or specifically phosphorylated S129-aSN (paSN). β-actin (βAc) levels were measured as loading control and for normalization. For reference, LRRK2 levels detected via immunoblot are shown comparing single and double-transgenics. Different α-synuclein protein species/forms are marked as follows: mo, monomer; ol, oligomer; tr, truncated. For reference, in the upper panels the performance and specificity of the antibodies are illustrated in the two right lanes comparing WT and KO (aSN knock-out) brain samples and were added to indicate unspecific cross-reactive proteins (taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036581#pone.0036581.s007" target="_blank">Figure S7</a>). Graphs represent quantifications of monomeric aSN and paSN/aSN, all normalized to βAc. Circles represent individual mice, the means are indicated as horizontal bars and % are normalized to the levels in haSN(A53T) single transgenics. p-values were determined by two-tailed, unequal variances Student’s t-test. Genotypes: aSN = haSN(A53T), aSN/LRRK2 = haSN(A53T)/hLRRK2(G2019S), Ntg = non-transgenic wildtype littermate control and KO = aSN knock-out mice.</p

Motor assessment and aSN/Tau protein characterization in hLRRK2(G2019S) mice.

<p>(A) Motor skill learning of 4-month-old male and 6-month-old female hLRRK2(G2019S) and Ntg controls in the 3-step accelerated rotarod task over four consecutive days. The number of mice per genotype is indicated. Three batches of animals were included in this graph (single transgenic and Ntg animals from experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036581#pone-0036581-g003" target="_blank">Figure 3B</a> as well as a separate batch). p-values were determined by repeated measure ANOVA (group effect males: F(1,119) = 9.42, p = 0.003, group effect females: F(1,52) = 3.74, p = 0.059). (B) Novelty-induced horizontal locomotor activity of 7.3- and 28.2-month-old hLRRK2(G2019S) and Ntg mice. Bar graphs show the sum of the distance travelled from 5–30 min and from 35–60 min. The number of mice per genotype is indicated. p-values were determined either by repeated measure ANOVA (group effect males 7.3 M: F(1,16) = 4.044, p = 0.061; group effect males 28.2 M: F(1,16) = 0.093, p = 0.764) or by two-tailed, unequal variances Student’s t-test. (C) Western blotting of forebrain homogenates from 15-month-old hLRRK2(G2019S) (TG) and Ntg male mice. Lower panel: Shown are levels of mouse α-synuclein (aSN) and phospho-α-synuclein Ser129 (paSN) as well as mouse microtubule-associated protein Tau and phospho-Tau Ser202/Thr205 (pTau). β-actin (βAc) was used as loading control and for normalization. Upper panel shows the results of the immunoblot quantifications. Circles represent individual mice, the means (% normalized to Ntg) are indicated as horizontal bars. p-values were determined by two-tailed, unequal variances Student’s t-test. Ntg: non-transgenic wildtype littermate control.</p