(A) Enzymatic removal of PSA-NCAM with EndoN i.c.v. infusion 24 h before training disrupts water maze acquisition on days 2 and 3 (p<0.05), and post-training (on day 1 and 2) plannexin injection restores the performance of EndoN pre-treated rats to control levels (##p<0.01, EndoN/Plannexin vs. EndoN/Control; *p<0.05 EndoN/plannexin vs. Vehicle control; p<0.01 EndoN/Control vs Vehicle Control, N = 9/group).

<p>(B) In the probe trial on day 3, no significant differences were detected between the three treatment groups. N = 9/group. (C)EndoN induces cleavage of PSA- NCAM in the dentate gyrus. Data are expressed as a percentage of the control group (vehicle-injected), **p<0.01 versus control group. N = 5–6/group. (D) PST mice, showing a genetic deficit in PSA-NCAM expression, are impaired in water maze learning (^αp<0.05 vs trial 1). Plannexin treatment (arrows) restored water maze learning in PST mice (PST/Control vs. WT/Control ** <i>p</i><0.01, PST/Plannexin vs PST/Control ## <i>p</i><0.01 and PST/Plannexin vs. WT/control n.s.). N = 11–17/group. (E) In the probe trial on day 3, no significant differences were detected between PST and WT littermates or between PST control and PST plannexin-treated subjects. N = 10–11/group. Results are expressed as mean ± SEM.</p

(A) Plannexin i.c.v. infusion immediately after the first and second training session (arrows) improves water maze learning (A, * <i>p</i><0.05).

<p>(B) In the probe trial on day 3, rats showed better memory retention for the platform location compared to the control group (* <i>p</i><0.05). (<b>C</b>) A scramble peptide infused i.c.v. immediately after the first and second training session (arrows) does not alter water maze learning as compared to vehicle-treated rats. (D) In the probe trial on day 3, rats infused i.c.v with a scramble peptide showed similar memory retention for the platform location compared to the vehicle group. N = 10/group. (<b>E</b>) Plannexin infusion did not affect a hippocampus-independent memory task, acoustic fear conditioning. No difference was detected in freezing levels between plannexin and control groups during the post-shock period at training and during tone presentation of the 24 h test. N = 8/group. Results are expressed as mean ± SEM.</p

Localization of the plannexin sequence in the NCAM Ig module.

<p>A space-filling model of Ig2 with two 180° rotation projections (margenta) is shown (PDB 1QZ1). The sequence of plannexin is mapped in yellow. The figure was made using PyMOL Molecular Viewer (DeLano Scientific LLC. San Francisco, CA, USA).</p

Plannexin enhances synaptic transmission of the Schaffer collateral pathway.

<p>A) Bath application of 10 µg/ml (i.e., 1.94 µM) plannexin increased the slope of the fEPSP evoked by Schaffer collateral stimulation (n = 17). B) A scrambled variant of plannexin did not affect the fEPSP (n = 5). C) Plannexin did not modulate the slope of the fEPSPs evoked by perforant pathway stimulation (n = 8). D) Summary of A–C, ** <i>p</i><0.01.</p

Synaptic expression of glutamate receptor subunits after plannexin treatment and water maze training: (A) GluA1 subunit; (B) GluA2 subunit; (C) GluA3 subunit; (D) GluN1B.

<p>Rats received 1 daily i.c.v. infusion of either vehicle or plannexin over 2 consecutive days either under basal conditions (untrained) or immediately after training in the water maze on days 1 and 2 (trained). Hippocampal samples were taken 2 days after the last drug infusion either under basal conditions (untrained) or following a 3^rd spatial training day and a probe trial just before sacrifice on day 4 (trained). Data are the mean ± SEM. *<i>p</i><0.05 vs. controls. C = Control; P = Plannexin.</p

The effect of EndoN treatment on plannexin-induced neurite outgrowth and survival.

<p>(a) 125,000 cells/cm² CGNs were left to differentiate for six days in the presence of high potassium (40 mM), followed by a 48 h incubation period with or without 15 nM EndoN. Cultures were double immunostained for GAP-43 (green) and PSA-NCAM (red). 12,500 cells/cm² Hippocampal neurons (b) and CGNs (c) were grown for 24 h in the presence or absence of 1.74 µM plannexin and treated with 30 nM EndoN (<b>b</b>) or 15 nM EndoN (<b>c</b>). Results from 4–5 experiments are expressed as a percentage ± SEM, with unstimulated controls set at 100%. ***<i>p</i><0.001 vs. untreated controls; ⁺⁺⁺<i>p</i><0.001 vs. peptide-treated cultures without EndoN treatment.</p

FGL triggers hippocampal FGFR1 phosphorylation in vitro and in vivo.

<p>(A) Cartoon structure of the double fibronectin module (FN1+FN2) of human NCAM (Protein Data Bank number 2VKW). The FGL sequence is shown in red with the two glutamine residues critical for the binding to the FGF-receptor highlighted in magenta. (B) Top: Representative immunoblot showing the in vitro phosphorylation of FGFR1 after stimulation of Trex293 cells that express Strep-tagged human FGFR1 with different concentrations of FGL and 10 ng/ml FGF1 (positive control) for 20 min. Bottom: Quantification of FGFR1 phosphorylation by FGL was performed by densitometric analysis of band intensity from four independent experiments similar to the one shown in the upper panel. (C) Phosphorylation of FGFR1 and TrkB was examined from hippocampal homogenates with an enzyme-linked immunosorbent assay (ELISA) 1 h after FGL subcutaneous injection. <i>N</i>, number of animals. Results are expressed as percentage ± SEM, with untreated controls set at 0%. (D–F) Phosphorylation of PLCγ (D), Shc (E), and FRS2 (F) in vitro was examined by Western blot, as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001262#pbio-1001262-g001" target="_blank">Figure 1B</a>. Treatment with FGF1 served as the positive control. Results from four independent experiments are expressed as a percentage ± SEM, with untreated controls set at 100%. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 compared with controls. Statistics were carried out according to the <i>t</i> test.</p

FGL enhances spatial learning.

<p>(A) Mean distances swam to find the hidden platform in the Morris water maze are represented for control rats (white symbols) and FGL-treated rats (black symbols) over 2 training days (four trials each). <i>N</i>, number of animals. Statistical significance was analyzed with repeated-measures ANOVA. (B) Cumulative frequency distributions of the distances swam by individual rats. Each data point represents the distance swam by one rat in the last trial of each day.</p

FGL induces AMPA receptor synaptic delivery via PKC activation.

<p>(A) Left: CA1 pyramidal neurons that express GluA1-GFP (green) on a DAPI-stained (blue) organotypic slice culture, imaged with laser-scanning confocal microscopy. Bar = 50 µm. Right: High-magnification image of GluA1-GFP-expressing neurons. Bar = 20 µm. (B) Schematic diagram that presents whole-cell recordings obtained from a neuron expressing GluA1-GFP (infected, green) and an adjacent non-fluorescent (uninfected, white) neuron. (C) AMPAR-mediated responses were recorded at −60 mV and +40 mV. The rectification index was calculated as the ratio of responses at these holding potentials. The <i>p</i> value was determined using the Mann-Whitney test. (D–H) FGL-induced rectification after incubation with inhibitors of different signal transduction pathways: MEK, PD98059 (D); PI3K, LY294002 (E); PKC, chelerythrine (F); classical PKC isoforms, GF109203X (G); atypical PKC isoforms (H). Sample traces are shown above the corresponding columns of the plot. <i>N</i>, number of cells. The <i>p</i> value was determined using the Mann-Whitney test. Scale bars = 15 pA and 10 ms.</p

FGL-induced enhanced cognition depends on PKC activity.

<p>(A, B) Mean distances traveled to find the hidden platform in the Morris water maze are represented for control rats (white circles), FGL-treated rats (black circles), and rats treated with FGL and the PKC inhibitor (grey squares; A, chelerythrine; B, GF109203X), over the 2 training days (four trials each). <i>N</i>, number of animals. Statistical significance was analyzed with repeated-measures ANOVA followed by Bonferroni's post hoc test for individual trials. A: *<i>p</i><0.05, FGL+Veh compared to FGL+Chel and Veh/Chel groups. #<i>p</i><0.05, FGL+Veh compared to FGL+Chel but not compared to Veh/Chel. B: *<i>p</i><0.05, FGL+Veh compared to FGL+GF109203X and Veh/GF109203X groups. #<i>p</i><0.05, FGL+Veh compared to Veh/GF109203X but not compared to FGL+GF109203X. (C) Probe test. Average time spent in the target quadrant of the Morris water maze (where the hidden platform had been present during training) for control rats (white column), FGL-treated rats (black column), or rats treated with FGL plus chelerythrine (grey column). Statistical significance was calculated with Bonferroni's post hoc test.</p