Journal: PLoS Biology

Article Title: The Sphingolipid Receptor S1PR2 Is a Receptor for Nogo-A Repressing Synaptic Plasticity

doi: 10.1371/journal.pbio.1001763

Figure Lengend Snippet: (A) S1PR2 expression in the hippocampus. CA, cornu ammonis ; DG, dentate gyrus. (B) Magnification of the boxed region of CA1 depicted in (A). (C) S1PR2 expression in the cerebellum. GCL, granule cell layer; ML, molecular layer; PCL, Purkinje cell layer. (D) Magnification of the boxed region depicted in (C). (E) S1PR2 expression in the motor cortex. (F) Magnification of the boxed region depicted in (E). (G,H) S1PR2 expression in motoneuron cell bodies (arrows) and βIII-Tubulin-positive fibers (arrowheads) in the spinal cord. (I,J,K) S1PR2 expression in βIII-Tubulin-positive axons bundles (arrowheads) and cell bodies (arrows) of retinal ganglion cells. Scale bars: (A) 300 µm; (B) 30 µm; (C) 200 µm; (D) 15 µm; (E) 90 µm; (F) 30 µm; (G,H) 20 µm; (I–K) 15 µm.

Article Snippet: The following primary antibodies were used: β Tubulin (Chemicon, MAB3408; 1∶1,000), βIII Tubulin (Promega, G712A; 1∶1,000), β-Actin (Sigma, A5441; 1∶1,000), BrdU (AbD Serotec, function-blocking experiments: 5 µg/ml), DAPI (Invitrogen, D1306, 1∶1,000), EEA1 (Cell Signaling, 2411; 1∶100), GAPDH (Abcam, ab8245; 1∶20,000), HA (Roche, 11867423001, 1∶200), His (Santa Cruz, sc-804, 1∶500), Pan-CDH (Abcam, ab6528; 1∶1,000), Nogo-A (1∶10,000, ), Nogo-A (Rb173A/Laura, 1∶200), Nogo-A/B (Bianca, Rb1, 1: 20,000, ), Phalloidin-Alexa488 (Invitrogen; 1∶500), RhoA (Cell Signaling, 2117; 1∶1,000), S1PR2 (Imgenex, IMG-6135A; 1∶250), S1PR2 (AbD Serotec custom made HuCAL antibody AbD14533.1 addressing extracellular S1PR2 ECL2; WB 1∶1,000; IHC 1∶100; TEM 1∶100), S1PR2 (Santa Cruz, sc-365589; 1∶500), S1PR5 (Abcam, ab13130; 1∶500; function-blocking experiments: 5 µg/ml), sphingosine 1-phosphate (Funakoshi, 274594052; function-blocking experiments: 5 µg/ml), Ubiquitin (Enzo Life Sciences, UWO150; 1∶1,000).

Techniques: Expressing

Journal: PLoS Biology

Article Title: The Sphingolipid Receptor S1PR2 Is a Receptor for Nogo-A Repressing Synaptic Plasticity

doi: 10.1371/journal.pbio.1001763

Figure Lengend Snippet: (A) Schematic structure of Nogo-A showing the inhibitory domains Nogo-A-Δ20 (Δ20, orange), Nogo-66 (blue), and Nogo-A-ext. Transmembrane domains are indicated in dark grey. RHD, reticulon homology domain. (B) Nogo-A (∼200 kDa) co-immunoprecipitated with S1PR2 (∼40 kDa) and vice-versa in WT but not Nogo-A −/− or S1PR2 −/− brain extracts (BE). If specified, the following controls were used in WT BE instead of the IP antibody to confirm the specificity of the interaction: IgG, control antibody; Ctrl R, resin only control; qAbR, quenched antibody (Ab) resin control. Input loading control: β-Actin (∼42 kDa). (C) S1PR2 immunoprecipitated with His-tagged Δ20 but not heat-inactivated (hi) Δ20 in S1PR2-overexpressing membranes. Input loading control: S1PR2. (D) His-tagged Δ20 but not hi Δ20 immunoprecipitated with S1PR2 in S1PR2-overexpressing membranes. Input loading control: S1PR2. (E) Nogo-A-ext bound specifically to biosensor-immobilized S1PR2-overexpressing versus control membranes ( K D ∼142 nM). A Scatchard plot analysis is shown on the right. (F) 1 µM S1P does not modulate the interaction between Nogo-A-ext and S1PR2 when compared to the methanol (MeOH) vehicle control (MeOH, K D ∼192 nM; S1P, K D ∼202 nM). A Scatchard plot analysis is shown on the right. (G) Microscale thermophoresis binding analysis of Δ20 to S1PR2 extracellular domains: ECL2 ( K D ∼280 nM), ECL3 ( K D ∼350 nM), ECL1 ( K D ∼1.7 µM), and N-terminus ( K D ∼11 µM). Scrambled ECL1 (ECL1-scr) was used as control ( K D ∼17 µM). Arrows indicate the identified Δ20-binding loops in S1PR2. (H) Nogo-66 binding to S1PR2 extracellular domains is unspecific: ECL2 ( K D ∼7 µM), ECL1 ( K D ∼46 µM), ECL3 ( K D ∼67 µM). No binding to the N-Terminus or to ECL1-scr is observed.

Article Snippet: The following primary antibodies were used: β Tubulin (Chemicon, MAB3408; 1∶1,000), βIII Tubulin (Promega, G712A; 1∶1,000), β-Actin (Sigma, A5441; 1∶1,000), BrdU (AbD Serotec, function-blocking experiments: 5 µg/ml), DAPI (Invitrogen, D1306, 1∶1,000), EEA1 (Cell Signaling, 2411; 1∶100), GAPDH (Abcam, ab8245; 1∶20,000), HA (Roche, 11867423001, 1∶200), His (Santa Cruz, sc-804, 1∶500), Pan-CDH (Abcam, ab6528; 1∶1,000), Nogo-A (1∶10,000, ), Nogo-A (Rb173A/Laura, 1∶200), Nogo-A/B (Bianca, Rb1, 1: 20,000, ), Phalloidin-Alexa488 (Invitrogen; 1∶500), RhoA (Cell Signaling, 2117; 1∶1,000), S1PR2 (Imgenex, IMG-6135A; 1∶250), S1PR2 (AbD Serotec custom made HuCAL antibody AbD14533.1 addressing extracellular S1PR2 ECL2; WB 1∶1,000; IHC 1∶100; TEM 1∶100), S1PR2 (Santa Cruz, sc-365589; 1∶500), S1PR5 (Abcam, ab13130; 1∶500; function-blocking experiments: 5 µg/ml), sphingosine 1-phosphate (Funakoshi, 274594052; function-blocking experiments: 5 µg/ml), Ubiquitin (Enzo Life Sciences, UWO150; 1∶1,000).

Techniques: Immunoprecipitation, Microscale Thermophoresis, Binding Assay

Journal: PLoS Biology

Article Title: The Sphingolipid Receptor S1PR2 Is a Receptor for Nogo-A Repressing Synaptic Plasticity

doi: 10.1371/journal.pbio.1001763

Figure Lengend Snippet: (A) Representative confocal micrographs of 3T3 cells stained alive (Non-perm) or fixed (Perm) for S1PR2 before (control) and 30 min after Δ20 treatment at 37°C. (B) Mean fluorescence intensity quantification of the cell surface staining shown in (A). (C) Addition of Δ20 downregulates cell surface S1PR2 in 3T3 plasma membranes (PM): immunoblot and relative quantification thereof. Loading control: β-Actin. (D) Representative confocal micrographs of 3T3 cells incubated with 1 µM HA-tagged Δ20 for 1 h at 4°C (pulse), which were then subsequently chased for 15 and 30 min at 37°C. Cells were stained with an anti-HA (Δ20), S1PR2, or EEA1 antibody (early endosomes). Arrows indicate cell surface-bound Δ20 (top panel) or colocalization of Δ20 and S1PR2 in early endosomes (middle and bottom panel). The inset panel shows an enlarged view of the boxed region. (E) Western blot analysis of ubiquitinated and non-ubiquitinated protein fractions of 3T3 cells 30 min after Δ20 or S1P treatment. Data shown are means ± SEM ( n = 3–6 experiments; ** p< 0.01, *** p< 0.001). Scale bars: (A,D) 50 µm.

Article Snippet: The following primary antibodies were used: β Tubulin (Chemicon, MAB3408; 1∶1,000), βIII Tubulin (Promega, G712A; 1∶1,000), β-Actin (Sigma, A5441; 1∶1,000), BrdU (AbD Serotec, function-blocking experiments: 5 µg/ml), DAPI (Invitrogen, D1306, 1∶1,000), EEA1 (Cell Signaling, 2411; 1∶100), GAPDH (Abcam, ab8245; 1∶20,000), HA (Roche, 11867423001, 1∶200), His (Santa Cruz, sc-804, 1∶500), Pan-CDH (Abcam, ab6528; 1∶1,000), Nogo-A (1∶10,000, ), Nogo-A (Rb173A/Laura, 1∶200), Nogo-A/B (Bianca, Rb1, 1: 20,000, ), Phalloidin-Alexa488 (Invitrogen; 1∶500), RhoA (Cell Signaling, 2117; 1∶1,000), S1PR2 (Imgenex, IMG-6135A; 1∶250), S1PR2 (AbD Serotec custom made HuCAL antibody AbD14533.1 addressing extracellular S1PR2 ECL2; WB 1∶1,000; IHC 1∶100; TEM 1∶100), S1PR2 (Santa Cruz, sc-365589; 1∶500), S1PR5 (Abcam, ab13130; 1∶500; function-blocking experiments: 5 µg/ml), sphingosine 1-phosphate (Funakoshi, 274594052; function-blocking experiments: 5 µg/ml), Ubiquitin (Enzo Life Sciences, UWO150; 1∶1,000).

Techniques: Staining, Fluorescence, Western Blot, Incubation

Journal: PLoS Biology

Article Title: The Sphingolipid Receptor S1PR2 Is a Receptor for Nogo-A Repressing Synaptic Plasticity

doi: 10.1371/journal.pbio.1001763

Figure Lengend Snippet: (A,C) Representative pictures of 3T3 fibroblasts treated with JTE-013 or vehicle (DMSO) (A), or stably carrying a S1pr2 shRNA (sh- S1pr2 ) or empty vector (sh-Vec) construct (C) and plated on control, Nogo-A-Δ20 or myelin substrates. (B,D) Cell spreading quantification of (A) and (C). (E) Representative pictures of MEFs isolated from WT or S1PR2 −/− mice and plated on control, Nogo-A- Δ20, or myelin substrates. (F) Cell spreading quantification of (E). Cells were stained with Alexa488-conjugated Phalloidin in (A, C, and E). (G,I) Representative pictures of P5–8 cerebellar granule neurons treated with JTE-013 or DMSO (G), or isolated from S1PR2 −/− or WT mice (I) and plated on PLL (ctrl), Nogo-A-Δ20 or myelin substrates. (H,J) Normalized mean neurite length per cell quantification of (G) and (I). Neurons were stained with βIII-Tubulin in (G) and (I). Data shown are means ± SEM ( n = 3–6 experiments; * p< 0.05, ** p< 0.01, *** p< 0.001). Scale bars: 50 µM.

Article Snippet: The following primary antibodies were used: β Tubulin (Chemicon, MAB3408; 1∶1,000), βIII Tubulin (Promega, G712A; 1∶1,000), β-Actin (Sigma, A5441; 1∶1,000), BrdU (AbD Serotec, function-blocking experiments: 5 µg/ml), DAPI (Invitrogen, D1306, 1∶1,000), EEA1 (Cell Signaling, 2411; 1∶100), GAPDH (Abcam, ab8245; 1∶20,000), HA (Roche, 11867423001, 1∶200), His (Santa Cruz, sc-804, 1∶500), Pan-CDH (Abcam, ab6528; 1∶1,000), Nogo-A (1∶10,000, ), Nogo-A (Rb173A/Laura, 1∶200), Nogo-A/B (Bianca, Rb1, 1: 20,000, ), Phalloidin-Alexa488 (Invitrogen; 1∶500), RhoA (Cell Signaling, 2117; 1∶1,000), S1PR2 (Imgenex, IMG-6135A; 1∶250), S1PR2 (AbD Serotec custom made HuCAL antibody AbD14533.1 addressing extracellular S1PR2 ECL2; WB 1∶1,000; IHC 1∶100; TEM 1∶100), S1PR2 (Santa Cruz, sc-365589; 1∶500), S1PR5 (Abcam, ab13130; 1∶500; function-blocking experiments: 5 µg/ml), sphingosine 1-phosphate (Funakoshi, 274594052; function-blocking experiments: 5 µg/ml), Ubiquitin (Enzo Life Sciences, UWO150; 1∶1,000).

Techniques: Stable Transfection, shRNA, Plasmid Preparation, Construct, Isolation, Staining

Journal: PLoS Biology

Article Title: The Sphingolipid Receptor S1PR2 Is a Receptor for Nogo-A Repressing Synaptic Plasticity

doi: 10.1371/journal.pbio.1001763

Figure Lengend Snippet: (A) 3T3 cells transfected with siRNAs against G 12 , G 13 , G q , or Larg , or control (ctrl) siRNA were replated on a Nogo-A-Δ20 substrate and assessed for cell spreading. G i/o was blocked with Pertussis Toxin (PTX) for which saline was used as control. JTE-013 was co-applied to G 13 -siRNA-treated cells to investigate a cumulative effect. (B) Transfection of DIV4 E19 cortical neurons with siRNA against G 13 but not G 12 similarly rescued Nogo-A-Δ20-induced neurite outgrowth inhibition. (C,D) Nogo-A-Δ20-induced RhoA activation was assessed in JTE-013- versus DMSO-treated cells (C) or in cells carrying a stable knockdown of S1PR2 (sh- S1pr2 ) versus control vector (sh-Vec) (D). (E,F) Relative quantification of (C) and (D), respectively. (G,H) Competitive ELISA quantifications of extra- (EC) and intracellular (IC) S1P levels in 3T3 cells (G) and cerebellar granule neurons (H) before and after 30 and 60 min incubation with Nogo-A-Δ20. (I) Quantification of Nogo-A-Δ20-mediated cell spreading inhibition in the presence of the SphK-specific blocker D,L- threo -dihydrosphingosine (DHS) or in SphK1 −/− or SphK2 −/− MEFs. (J,K) 3T3 cells were plated on a Nogo-A-Δ20 substrate in the presence of the function blocking anti-S1P antibody Sphingomab (J) or of exogenous S1P (K) and assessed for cell spreading. Co-application of JTE-013 significantly reversed the modulatory effects obtained by S1P (K) but not anti-S1P (J). Anti-BrdU antibody or methanol was used as control in (J) and (K). Data shown are means ± SEM ( n = 3–6 experiments; * p< 0.05, ** p< 0.01, *** p< 0.001).

Article Snippet: The following primary antibodies were used: β Tubulin (Chemicon, MAB3408; 1∶1,000), βIII Tubulin (Promega, G712A; 1∶1,000), β-Actin (Sigma, A5441; 1∶1,000), BrdU (AbD Serotec, function-blocking experiments: 5 µg/ml), DAPI (Invitrogen, D1306, 1∶1,000), EEA1 (Cell Signaling, 2411; 1∶100), GAPDH (Abcam, ab8245; 1∶20,000), HA (Roche, 11867423001, 1∶200), His (Santa Cruz, sc-804, 1∶500), Pan-CDH (Abcam, ab6528; 1∶1,000), Nogo-A (1∶10,000, ), Nogo-A (Rb173A/Laura, 1∶200), Nogo-A/B (Bianca, Rb1, 1: 20,000, ), Phalloidin-Alexa488 (Invitrogen; 1∶500), RhoA (Cell Signaling, 2117; 1∶1,000), S1PR2 (Imgenex, IMG-6135A; 1∶250), S1PR2 (AbD Serotec custom made HuCAL antibody AbD14533.1 addressing extracellular S1PR2 ECL2; WB 1∶1,000; IHC 1∶100; TEM 1∶100), S1PR2 (Santa Cruz, sc-365589; 1∶500), S1PR5 (Abcam, ab13130; 1∶500; function-blocking experiments: 5 µg/ml), sphingosine 1-phosphate (Funakoshi, 274594052; function-blocking experiments: 5 µg/ml), Ubiquitin (Enzo Life Sciences, UWO150; 1∶1,000).

Techniques: Transfection, Inhibition, Activation Assay, Plasmid Preparation, Competitive ELISA, Incubation, Blocking Assay

Journal: PLoS Biology

Article Title: The Sphingolipid Receptor S1PR2 Is a Receptor for Nogo-A Repressing Synaptic Plasticity

doi: 10.1371/journal.pbio.1001763

Figure Lengend Snippet: (A,B) Hippocampal WT (A) and Nogo-A −/− (B) slices were treated with JTE-013 or vehicle (DMSO) (WT DMSO : n = 8; Nogo-A −/− DMSO : n = 10; WT JTE-013 : n = 11; Nogo-A −/− JTE-013 : n = 9). 60 min after theta-burst stimulation (arrow), a significant difference in LTP could be observed between JTE-013 and DMSO treatment in WT (A) but not Nogo-A −/− (B) slices. (C,D) Input-output strength revealed no differences in JTE-013- versus DMSO-treated slices of WT (C) and Nogo-A −/− (D) mice (WT DMSO : n = 6; Nogo-A −/− DMSO : n = 6; WT JTE-013 : n = 7; Nogo-A −/− JTE-013 : n = 6). (E,F) PPF revealed no alterations in JTE-013- versus DMSO-treated slices of WT (E) and Nogo-A −/− (F) mice (WT DMSO : n = 7; Nogo-A −/− DMSO : n = 6; WT JTE-013 : n = 5; Nogo-A −/− JTE-013 : n = 6). (G) LTP was measured upon simultaneous neutralization of S1PR2 using JTE-013 and of Nogo-A using 11c7 (IgG1 + DMSO: n = 7; IgG1 + JTE-013: n = 6; 11c7 + DMSO: n = 8; 11c7 + JTE-013: n = 6). (H) LTP was measured upon simultaneous neutralization of S1PR2 using JTE-013 and of NgR1 using anti-NgR1 (DMSO: n = 7; JTE-013: n = 9; anti-NgR1 + JTE-013: n = 8). (I) Rat motor forelimb area brain slices were treated with JTE-013 ( n = 7) or DMSO ( n = 8). Peak amplitudes were significantly larger in JTE-013- versus DMSO-treated slices upon repeated inductions of LTP (multiple arrows). (J) Input-output strength revealed no differences in JTE-013- ( n = 8) versus DMSO-treated ( n = 12) cortical slices. Insets show representative traces. Data shown are means ± SEM (* p< 0.05). n indicates the number of mice used.

Article Snippet: The following primary antibodies were used: β Tubulin (Chemicon, MAB3408; 1∶1,000), βIII Tubulin (Promega, G712A; 1∶1,000), β-Actin (Sigma, A5441; 1∶1,000), BrdU (AbD Serotec, function-blocking experiments: 5 µg/ml), DAPI (Invitrogen, D1306, 1∶1,000), EEA1 (Cell Signaling, 2411; 1∶100), GAPDH (Abcam, ab8245; 1∶20,000), HA (Roche, 11867423001, 1∶200), His (Santa Cruz, sc-804, 1∶500), Pan-CDH (Abcam, ab6528; 1∶1,000), Nogo-A (1∶10,000, ), Nogo-A (Rb173A/Laura, 1∶200), Nogo-A/B (Bianca, Rb1, 1: 20,000, ), Phalloidin-Alexa488 (Invitrogen; 1∶500), RhoA (Cell Signaling, 2117; 1∶1,000), S1PR2 (Imgenex, IMG-6135A; 1∶250), S1PR2 (AbD Serotec custom made HuCAL antibody AbD14533.1 addressing extracellular S1PR2 ECL2; WB 1∶1,000; IHC 1∶100; TEM 1∶100), S1PR2 (Santa Cruz, sc-365589; 1∶500), S1PR5 (Abcam, ab13130; 1∶500; function-blocking experiments: 5 µg/ml), sphingosine 1-phosphate (Funakoshi, 274594052; function-blocking experiments: 5 µg/ml), Ubiquitin (Enzo Life Sciences, UWO150; 1∶1,000).

Techniques: Neutralization