Journal: Progress in neurobiology

Article Title: Intracellular accumulation of tau oligomers in astrocytes and their synaptotoxic action rely on Amyloid Precursor Protein Intracellular Domain-dependent expression of Glypican-4

doi: 10.1016/j.pneurobio.2023.102482

Figure Lengend Snippet: (A1, B1) Representative examples of oTau internalization in cultured hippocampal astrocytes treated for 24 hours with vehicle (A) or anti-GPC4 antibody (B) before 1-hour incubation with 200 nM ex-oTau in the culture medium. Tau oligomers were labelled with the IRIS-5 fluorophore (oTauIRIS-5). (A2, B2) Panels showing the “internalization maps”, where the black dots represent points in which the fluorescence of GFAP and oTauIRIS-5 co-localizes, thus indicating oTau internalization. Ellipses in panels A and B indicate clusters of tau oligomers that i) are internalized (green) or (ii) remained attached to the plasma membrane of astrocytes without enter (blue). The presence of green dots in panel B1 (oTauIRIS-5-GFAP) that are not paralleled by black dots in the corresponding internalization map (panel B2) indicate tau oligomers that are attached to the membrane but not (yet) internalized. Scale bars: 10 μm in A1-B1. (C) Bar graph showing Internalization Index (see Piacentini et al., 2017) for oTau, after 1-h incubation in the presence of vehicle, or 24-h pretreatment with anti GPC4. (n=10 independent fields by three independent experiments for both conditions). ***p<0.001.

Article Snippet: Cells were incubated overnight at 4 °C with an appropriate combination of the following antibodies, diluted in the same blocking solution: mouse anti-GFAP (#3670, 1:500, Cell Signaling Technology, Danvers, MA) or rabbit anti-GFAP (#12389, 1:500, Cell Signaling), and mouse anti-Heparan Sulfate (10E4 epitope, USbiological) or rabbit anti-GPC4 (bs-2159R, 1:300, Bioss, Boston, MA); mouse anti-MAP2 antibody (1:300, Sigma); rabbit anti-Synapsin-1 (#5297, 1:300, Cell Signaling).

Techniques: Cell Culture, Incubation, Fluorescence, Membrane

Journal: Progress in neurobiology

Article Title: Intracellular accumulation of tau oligomers in astrocytes and their synaptotoxic action rely on Amyloid Precursor Protein Intracellular Domain-dependent expression of Glypican-4

doi: 10.1016/j.pneurobio.2023.102482

Figure Lengend Snippet: (A-B) Representative confocal images of a hippocampal section from WT (panel A) and APP KO (Panel B) immunolabeled for GFAP (red) and GPC4 (green). DAPI was used to stain cell nuclei. Scale bar 10 μm. (C) Representative Western blot analysis carried out on hippocampal lysates obtained from WT and APP KO mouse brain probed with anti GPC4 antibody (n=9 hippocampi for condition). (D) Bar graph quantifying the expression levels of GPC4 in experiments reported in panel C. (E) Bar graph showing relative expression of Gpc4 mRNA evaluated in cultured astrocytes from WT and APP KO mice (n=3 independent experiments for both). (F-G) Representative confocal images of GPC4 immunoreactivity in cultured astrocytes treated for 48 hours with vehicle (DMSO) or the γ secretase inhibitor X. Panels on the right represents GPC4 immunoreactivity only in the two conditions analyzed. Scale bar 10 μm. (H) Bar graph quantifying the expression levels of GPC4 in experiments reported in panel E-F. (I) Representative Western blot analysis carried out on lysated of cultured WT astrocytes treated for 48 hours with the inhibitor of the γ secretase ENLD006. (J) Bar graph quantifying the expression levels of GPC4 in experiments reported in panel I. (K) Bar graph showing the results of ChIP experiments demonstrating the binding of AICD to the Gpc4 promoter (aAICD: antibody anti-AICD; no ab: no antibody). *p<0.05.

Article Snippet: Cells were incubated overnight at 4 °C with an appropriate combination of the following antibodies, diluted in the same blocking solution: mouse anti-GFAP (#3670, 1:500, Cell Signaling Technology, Danvers, MA) or rabbit anti-GFAP (#12389, 1:500, Cell Signaling), and mouse anti-Heparan Sulfate (10E4 epitope, USbiological) or rabbit anti-GPC4 (bs-2159R, 1:300, Bioss, Boston, MA); mouse anti-MAP2 antibody (1:300, Sigma); rabbit anti-Synapsin-1 (#5297, 1:300, Cell Signaling).

Techniques: Immunolabeling, Staining, Western Blot, Expressing, Cell Culture, Binding Assay

Journal: Progress in neurobiology

Article Title: Intracellular accumulation of tau oligomers in astrocytes and their synaptotoxic action rely on Amyloid Precursor Protein Intracellular Domain-dependent expression of Glypican-4

doi: 10.1016/j.pneurobio.2023.102482

Figure Lengend Snippet: (A) Bar graphs showing the mean amplitude at peak (filled bars, left) of ATP (10 s, 100 μM)-induced intracellular Ca2+ transients in astrocytes treated for 1 hour with vehicle (n=254 studied cells) 200 nM ex-oTau (n=770) and ex-oTau after 24-h pretreatment with anti-GPC4 (n=570). (B) Faded bars show the mean number of spontaneous Ca2+ transients/min elicited after ATP stimulation in the above-mentioned conditions. (C) Bar graph showing the amount of ATP released by astrocytes in the culture medium in the following conditions: 1-h application of extracellular oligomeric tau after (n=6 independent experiments), or not (n=10), 24-h anti-GPC4 treatment. *p<0.05; **p<0.01; ***p<0.001; #p<0.05 and ###p<0.001.

Article Snippet: Cells were incubated overnight at 4 °C with an appropriate combination of the following antibodies, diluted in the same blocking solution: mouse anti-GFAP (#3670, 1:500, Cell Signaling Technology, Danvers, MA) or rabbit anti-GFAP (#12389, 1:500, Cell Signaling), and mouse anti-Heparan Sulfate (10E4 epitope, USbiological) or rabbit anti-GPC4 (bs-2159R, 1:300, Bioss, Boston, MA); mouse anti-MAP2 antibody (1:300, Sigma); rabbit anti-Synapsin-1 (#5297, 1:300, Cell Signaling).

Techniques:

Journal: Progress in neurobiology

Article Title: Intracellular accumulation of tau oligomers in astrocytes and their synaptotoxic action rely on Amyloid Precursor Protein Intracellular Domain-dependent expression of Glypican-4

doi: 10.1016/j.pneurobio.2023.102482

Figure Lengend Snippet: (A) Mean time course of FM1–43 intensity following 30 mM KCl stimulation in hippocampal neurons co-cultured with astrocytes and treated for 1 hour with either vehicle or ex-oTau (200 nM) after 24-h pre-treatment with vehicle or anti-GPC4 (aGPC4). (B) Curves obtained by the fitting of the main curves in A with the equation used to evaluate the single parameters (A and τ) for each studied condition. n=28 neurons analyzed for vehicle/vehicle; n=18 for vehicle/ex-oTau; n=29 for vehicle/aGPC4; n=61 for aGPC4/ex-oTau. (C1–3) Representative confocal images of immunoreactivity of cultured murine hippocampal neurons (MAP2-positive) for Synapsin-1 in the following conditions: vehicle-treated, 1-h ex-oTau treatment, 1-h ex-oTau treatment following 24 h lasting cell incubation with anti-GPC4. DAPI was used to label cell nuclei. Scale bar: 10 μm. (D) Bar graph quantifying data of experiments represented in C1–3. (E) Representative Western blot analysis carried-out on lysates from organotypic hippocampal slices treated as in panels C1–3. (F) Bar graph showing densitometric analysis of experiments reported in panel E. *p<0.05 and ***p<0.001; # p<0.05 and ###p<0.001. n.s., not significant difference.

Article Snippet: Cells were incubated overnight at 4 °C with an appropriate combination of the following antibodies, diluted in the same blocking solution: mouse anti-GFAP (#3670, 1:500, Cell Signaling Technology, Danvers, MA) or rabbit anti-GFAP (#12389, 1:500, Cell Signaling), and mouse anti-Heparan Sulfate (10E4 epitope, USbiological) or rabbit anti-GPC4 (bs-2159R, 1:300, Bioss, Boston, MA); mouse anti-MAP2 antibody (1:300, Sigma); rabbit anti-Synapsin-1 (#5297, 1:300, Cell Signaling).

Techniques: Cell Culture, Incubation, Western Blot

Journal: Progress in neurobiology

Article Title: Intracellular accumulation of tau oligomers in astrocytes and their synaptotoxic action rely on Amyloid Precursor Protein Intracellular Domain-dependent expression of Glypican-4

doi: 10.1016/j.pneurobio.2023.102482

Figure Lengend Snippet: (A-B) Representative confocal images of hippocampal section from WT mice (panel A) and the APPTA transgenic mice in which Threonine 668 of APP was substituted with non-phosphorylatable Alanine (Panel B), immunolabeled for GFAP (red) and GPC4 (green). DAPI was used to stain cell nuclei. Scale bar 75 μm. (C) Representative Western blot analysis carried out on hippocampal lysates obtained from WT and APPTA mice probed with anti GPC4 antibody (n=3 independent experiments). (D) Bar graph quantifying the expression levels of GPC4 in experiments reported in panel C (n=3 independent experiments). (E) Bar graph showing relative expression of Gpc4 mRNA evaluated in cultured astrocytes from WT and APPTA mice (n=3 independent experiments for both). *p<0.05.

Article Snippet: Cells were incubated overnight at 4 °C with an appropriate combination of the following antibodies, diluted in the same blocking solution: mouse anti-GFAP (#3670, 1:500, Cell Signaling Technology, Danvers, MA) or rabbit anti-GFAP (#12389, 1:500, Cell Signaling), and mouse anti-Heparan Sulfate (10E4 epitope, USbiological) or rabbit anti-GPC4 (bs-2159R, 1:300, Bioss, Boston, MA); mouse anti-MAP2 antibody (1:300, Sigma); rabbit anti-Synapsin-1 (#5297, 1:300, Cell Signaling).

Techniques: Transgenic Assay, Immunolabeling, Staining, Western Blot, Expressing, Cell Culture

Journal: Progress in neurobiology

Article Title: Intracellular accumulation of tau oligomers in astrocytes and their synaptotoxic action rely on Amyloid Precursor Protein Intracellular Domain-dependent expression of Glypican-4

doi: 10.1016/j.pneurobio.2023.102482

Figure Lengend Snippet: (A) Time-course of excitatory post-synaptic currents (EPSCs) recorded in organotypic hippocampal slices at CA1 after pairing stimuli to CA3. Curves represent EPSCs in slices treated with vehicle (black dots; n=10), 1-h ex-oTau (200 nM) alone (red dots; n=10) or after 24-h incubation with anti-GPC4 (green dots; n=10). Treatment with antibody alone is shown in yellow (n=10). Slices were obtained from 3 independent preparations (4 mice/preparation). (B) Bar graph indicating the mean values of LTP evaluated in the last 5 min of EPSC recording, *p<0.05; n.s.: not significant difference.

Article Snippet: Cells were incubated overnight at 4 °C with an appropriate combination of the following antibodies, diluted in the same blocking solution: mouse anti-GFAP (#3670, 1:500, Cell Signaling Technology, Danvers, MA) or rabbit anti-GFAP (#12389, 1:500, Cell Signaling), and mouse anti-Heparan Sulfate (10E4 epitope, USbiological) or rabbit anti-GPC4 (bs-2159R, 1:300, Bioss, Boston, MA); mouse anti-MAP2 antibody (1:300, Sigma); rabbit anti-Synapsin-1 (#5297, 1:300, Cell Signaling).

Techniques: Incubation

Journal: Neuron

Article Title: Unbiased Discovery of Glypican as a Novel Receptor for LRRTM4 in Regulating Excitatory Synapse Development

doi: 10.1016/j.neuron.2013.06.049

Figure Lengend Snippet: (A and B) In situ hybridizations showing expression of LRRTM2 (A) and LRRTM4 (B) in horizontal rat brain sections (arrowheads indicate DG). (C) Coomassie-stained SDS-PAGE gel of purified recombinant LRRTM2- and LRRTM4-ecto-Fc proteins. (D) Identification of glypicans as candidate LRRTM4 interactors by tandem mass spectrometry. LRRTM2-Fc and LRRTM4-Fc proteins were used as bait and Triton X-100-solubilized whole rat brain homogenate was used as prey. Neurexins (black bars) are the major surface protein identified by LRRTM2-Fc; glypicans (grey bars) are the major surface proteins identified by LRRTM4-Fc. Bar graph shows total number of spectra in which the identified proteins were found. (E and F) Cell surface binding assays. LRRTM2-Fc (red) shows no detectable binding to HEK293T cells expressing HA-tagged glypicans (green) (E). LRRTM4-Fc strongly binds to glypicans (F). Both bind FLAG-Nrx1ß(−S4). (G) Coomassie-stained SDS-PAGE gel of purified recombinant GPC4-Fc protein. (H) Identification of LRRTM3 and LRRTM4 in GPC4-Fc affinity purification of Triton X-100-solubilized crude synaptosomes. (I and J) Binding assays. GPC4-Fc (red) does not bind to myc-LRRTM2 (green) (I), but does bind to myc-LRRTM4 (J). See also Figure S1. Scale bar in (E, F, I, J) 10 μm.

Article Snippet: Primary antibodies were: goat anti-GFP (Abcam), mouse anti-PSD-95 (Thermo Scientific/Affinity BioReagents), mouse anti-Prox1 (Millipore), guinea pig anti-VGlut1 (Millipore), mouse anti-gephyrin and guinea pig anti-VGAT (Synaptic Systems, Goettingen, Germany), mouse anti-LRRTM4 (clone N205B/22, UCDavis/NIH Neuromab, CA, USA), sheep anti-LRRTM4 (R&D Systems), rabbit anti-GPC4 (aa 88-101; Immundiagnostik), rabbit anti-GluR1 (Calbiochem), rabbit anti-synapsin (Millipore), chicken anti-MAP2 (Sigma), mouse anti-myc 9E10 (Santa Cruz Biotechnology), mouse anti-HA (Covance), mouse anti-FLAG M2 (Sigma), mouse anti-heparan sulfate delta (3G10 epitope) (USBiological).

Techniques: In Situ, Expressing, Staining, SDS Page, Purification, Recombinant, Mass Spectrometry, Binding Assay, Affinity Purification

Journal: Neuron

Article Title: Unbiased Discovery of Glypican as a Novel Receptor for LRRTM4 in Regulating Excitatory Synapse Development

doi: 10.1016/j.neuron.2013.06.049

Figure Lengend Snippet: (A) Direct interaction of recombinant His-tagged ecto-LRRTM4 with GPC4-Fc. Fc, GPC4-Fc, Nrx1ß(−S4)-Fc or LPHN3-Fc were mixed with His-LRRTM4, precipitated and analyzed by Western blot. His-LRRTM4 binds to GPC4-Fc and Nrx1ß(−S4)-Fc, but not to Fc or LPHN3-Fc. (B) LRRTM4 bound to Nrx1ß(−S4) cannot simultaneously bind to GPC4. Recombinant HA-GPC4, Nrx1ß(−S4)-Fc and His-LRRTM4 or His-FLRT3 were mixed and precipitated with protein A/G agarose. Proteins bound to Nrx1ß(−S4)-Fc were analyzed with His and HA antibodies. Blot shows full-length GPC4. (C) LRRTM4 bound to GPC4 cannot simultaneously bind to Nrx1ß(−S4). HA-GPC4, Nrx1ß(−S4)-Fc and His-LRRTM4 or His-FLRT3 were mixed and precipitated with HA affinity matrix. Proteins bound to HA-GPC4 were analyzed with His and Fc antibodies. (D) Cell surface binding assays. LRRTM4-Fc (red) binding to HA-GPC4 (green) expressing HEK293T cells is strongly reduced in the presence of 0.5 mg/ml HS or following treatment with 1 U/ml heparinase (hep) III. HS and hepIII abolish background binding of LRRTM4-Fc to cells expressing vector alone. (E) Quantification of assays in (D). Bar graph shows mean ± SEM; a.u., arbitrary units. Only LRRTM4-Fc binding to HA-GPC4 + vehicle was significantly above the pDisplay + vehicle control condition (***p<0.001, post-hoc analysis using Bonferroni Multiple Comparisons test; all other comparisons p>0.05; n=11-29 cells per condition). (F) Schematic representation of HA-GPC4 glycosylation mutants. Green dots indicate proteolytic cleavage site; serine residues serving as HS GAG attachment sites (red dots) were mutated to alanine (blue dots). Mutation of all three GAG attachment sites strongly reduces binding of LRRTM4-Fc (red) to HA-GPC4 (green). See also Figure S2. Scale bar in (D, F) 10 μm.

Article Snippet: Primary antibodies were: goat anti-GFP (Abcam), mouse anti-PSD-95 (Thermo Scientific/Affinity BioReagents), mouse anti-Prox1 (Millipore), guinea pig anti-VGlut1 (Millipore), mouse anti-gephyrin and guinea pig anti-VGAT (Synaptic Systems, Goettingen, Germany), mouse anti-LRRTM4 (clone N205B/22, UCDavis/NIH Neuromab, CA, USA), sheep anti-LRRTM4 (R&D Systems), rabbit anti-GPC4 (aa 88-101; Immundiagnostik), rabbit anti-GluR1 (Calbiochem), rabbit anti-synapsin (Millipore), chicken anti-MAP2 (Sigma), mouse anti-myc 9E10 (Santa Cruz Biotechnology), mouse anti-HA (Covance), mouse anti-FLAG M2 (Sigma), mouse anti-heparan sulfate delta (3G10 epitope) (USBiological).

Techniques: Recombinant, Western Blot, Binding Assay, Expressing, Plasmid Preparation, Mutagenesis

Journal: Neuron

Article Title: Unbiased Discovery of Glypican as a Novel Receptor for LRRTM4 in Regulating Excitatory Synapse Development

doi: 10.1016/j.neuron.2013.06.049

Figure Lengend Snippet: (A) In situ hybridization showing LRRTM4 expression in a sagittal section of P14 rat hippocampus. LRRTM4 expression is limited to DG granule cells. (B) LRRTM4 protein (red) localizes to granule cell bodies and the DG molecular layer in P15 hippocampus (arrowheads). The presynaptic excitatory marker VGlut1 (blue) visualizes hippocampal architecture. (C, D) Postsynaptic localization of LRRTM4 in DIV14 hippocampal neurons. (C) LRRTM4-positive puncta (red) partially overlap with VGlut1 puncta (green). (D) LRRTM4 puncta (red) colocalize with the excitatory postsynaptic marker GluR1 (green). (E) In situ hybridization showing GPC4 expression in a sagittal section of P14 hippocampus. GPC4 is strongly expressed in DG and CA1, and weakly in CA3. (F) GPC4 protein (red) localizes to DG and CA1 cell bodies and neuropil in P21 hippocampus. Arrowheads indicate strong GPC4 staining in CA3 stratum lucidum. (G) Presynaptic localization of GPC4 in DIV16 hippocampal neuron. GPC4-positive puncta colocalize with VGlut1 (red) and are juxtaposed to puncta positive for the excitatory postsynaptic marker PSD-95 (blue). See also Figure S3. Scale bar in (A, B, E, F) 200 μm; in (C, D, G) 10 μm.

Article Snippet: Primary antibodies were: goat anti-GFP (Abcam), mouse anti-PSD-95 (Thermo Scientific/Affinity BioReagents), mouse anti-Prox1 (Millipore), guinea pig anti-VGlut1 (Millipore), mouse anti-gephyrin and guinea pig anti-VGAT (Synaptic Systems, Goettingen, Germany), mouse anti-LRRTM4 (clone N205B/22, UCDavis/NIH Neuromab, CA, USA), sheep anti-LRRTM4 (R&D Systems), rabbit anti-GPC4 (aa 88-101; Immundiagnostik), rabbit anti-GluR1 (Calbiochem), rabbit anti-synapsin (Millipore), chicken anti-MAP2 (Sigma), mouse anti-myc 9E10 (Santa Cruz Biotechnology), mouse anti-HA (Covance), mouse anti-FLAG M2 (Sigma), mouse anti-heparan sulfate delta (3G10 epitope) (USBiological).

Techniques: In Situ Hybridization, Expressing, Marker, Staining

Journal: Neuron

Article Title: Unbiased Discovery of Glypican as a Novel Receptor for LRRTM4 in Regulating Excitatory Synapse Development

doi: 10.1016/j.neuron.2013.06.049

Figure Lengend Snippet: (A-H) Coculture assays. (A) Myc-LRRTM4 (green) expressed in HEK293T cells cocultured with DIV7-8 hippocampal neurons induces clustering of the presynaptic excitatory marker VGlut1 (red) but not of the presynaptic inhibitory marker VGAT (red). (B) Quantification of assays in (A). LRRTM4 significantly increases the fractional VGlut1 area (area occupied by VGlut1 staining per myc-labeled surface area normalized to GFP-expressing control cells) compared to GFP cells (VGlut1: GFP 1.00 ± 0.32 (n=30 cells) vs. LRRTM4 14.18 ± 2.54 (n=27); ***p<0.0001, Mann-Whitney test. VGAT: GFP 1.00 ± 0.22 (n=21 cells) vs. LRRTM4 2.07 ± 0.54 (n=23); n.s. p=0.1103 Mann-Whitney test). (C) Myc-LRRTM4 induces clustering of endogenous GPC4 (red). (D) Quantification of (C); GFP 1.00 ± 0.34 (n=19 cells) vs. LRRTM4 22.67 ± 4.52 (n=24); ***p<0.0001, Mann-Whitney test. (E) HA-GPC4 expressed in 293T cells induces clustering of the excitatory postsynaptic marker PSD-95 (red; arrowheads) but not of the inhibitory postsynaptic marker gephyrin (red). (F) Quantification of (E); PSD-95: GFP 1.00 ± 0.21 (n=18) vs. HA-GPC4 5.30 ± 0.58 (n=26); ***p<0.0001, Student’s t-test. Gephyrin: GFP 1.00 ± 0.23 (n=20) vs. HA-GPC4 1.96 ± 0.81 (n=25); n.s. p=0.7441, Student’s t-test. (G) HAGPC4-mediated clustering of endogenous LRRTM4 (red) in contacting dendrites requires GAG chains. HA-GPC4-mediated LRRTM4 clustering (arrowheads) is absent around HA-GPC4 AAA expressing cells. (H) Quantification of (G); GFP 1.00 ± 0.22 (n=53), HA-GPC4 15.06 ± 3.07 (n=78), HA-GPC4 AAA 2.17 ± 0.52 (n=53); ***p<0.001, Kruskal-Wallis test, Dunn’s multiple comparisons post-hoc test. Bar graphs show mean ± SEM. See also Figure S4. Scale bar in (A, C, E, G) 10 μm.

Article Snippet: Primary antibodies were: goat anti-GFP (Abcam), mouse anti-PSD-95 (Thermo Scientific/Affinity BioReagents), mouse anti-Prox1 (Millipore), guinea pig anti-VGlut1 (Millipore), mouse anti-gephyrin and guinea pig anti-VGAT (Synaptic Systems, Goettingen, Germany), mouse anti-LRRTM4 (clone N205B/22, UCDavis/NIH Neuromab, CA, USA), sheep anti-LRRTM4 (R&D Systems), rabbit anti-GPC4 (aa 88-101; Immundiagnostik), rabbit anti-GluR1 (Calbiochem), rabbit anti-synapsin (Millipore), chicken anti-MAP2 (Sigma), mouse anti-myc 9E10 (Santa Cruz Biotechnology), mouse anti-HA (Covance), mouse anti-FLAG M2 (Sigma), mouse anti-heparan sulfate delta (3G10 epitope) (USBiological).

Techniques: Marker, Staining, Labeling, Expressing, MANN-WHITNEY

Journal: Neuron

Article Title: Unbiased Discovery of Glypican as a Novel Receptor for LRRTM4 in Regulating Excitatory Synapse Development

doi: 10.1016/j.neuron.2013.06.049

Figure Lengend Snippet: (A-P) Coculture assays. (A) Myc-LRRTM2-induced clustering of synapsin puncta on the HEK293T cell surface in vehicle- or HS (0.5 mg/ml)-treated cocultures. (B) Quantification of synapsin immunoreactivity as a measure of presynaptic differentiation in assays in (A); vehicle 1.00 ± 0.14 (n=29 cells) vs. HS 1.14 ± 0.22 (n=30); p=0.9784, Mann-Whitney test. (C) HS blocks myc-LRRTM4-induced presynaptic differentiation. (D) Quantification of (C); vehicle 1.00 ± 0.13 (n=31) vs. HS 0.14 ± 0.03 (n=34); ***p<0.0001, Mann-Whitney test. (E) Myc-LRRTM2-induced presynaptic differentiation in vehicle- or hepIII (1 U/ml)-treated cultures. (F) Quantification of (E); vehicle 1.00 ± 0.09 (n=25) vs. hepIII 0.91 ± 0.09 (n=25); p=0.4676, Student’s t-test. (G) HepIII treatment strongly reduces myc-LRRTM4-induced presynaptic differentiation. (H) Quantification of (G); vehicle 1.00 ± 0.10 (n=27) vs. hepIII 0.23 ± 0.03 (n=35); ***p<0.0001, Student’s t-test. (I) Effect of Fc, GPC4-Fc and Nrx1ß(−S4)-Fc proteins (50 μg/ml) on LRRTM2-induced presynaptic differentiation. (J) Quantification of (I) (Fc 1.00 ± 0.11 (n=28), GPC4-Fc 1.05 ± 0.13 (n=27), Nrx1ß(−S4)-Fc 1.04 ± 0.10 (n=30); p=0.9511, ANOVA). (K) GPC4-Fc reduces myc-LRRTM4-induced presynaptic differentiation. (L) Quantification of (K); Fc 1.00 ± 0.10 (n=30), GPC4-Fc 0.30 ± 0.05 (n=33), Nrx1ß(−S4)-Fc 1.83 ± 0.27 (n=27); ***p<0.001; Fc vs. Nrx1ß(−S4)-Fc p>0.05; Kruskal-Wallis test, Dunn’s multiple comparison post-hoc test. (M) Neuronal GPC4 knockdown does not affect synapse formation onto LRRTM2-expressing 293T cells. Synapsin (syn), red; GFP, green; myc, blue. (N) Quantification of (M); control 1.00 ± 0.09 (n=19) vs. shGPC4 1.14 ± 0.24 (n=17); p=0.7934, Mann-Whitney test. (O) GPC4 knockdown decreases synapse formation onto LRRTM4-expressing cells. (P) Quantification of (O); control 1.00 ± 0.15 (n=24), shGPC4 0.47 ± 0.10 (n=30), rescue 0.96 ± 0.17 (n=32); **p<0.01, Kruskal-Wallis test, Dunn’s multiple comparison post-hoc test. See also Figure S6. Bar graphs show mean ± SEM. Scale bar in (A, C, E, G, I, K, M, O) 10 μm.

Article Snippet: Primary antibodies were: goat anti-GFP (Abcam), mouse anti-PSD-95 (Thermo Scientific/Affinity BioReagents), mouse anti-Prox1 (Millipore), guinea pig anti-VGlut1 (Millipore), mouse anti-gephyrin and guinea pig anti-VGAT (Synaptic Systems, Goettingen, Germany), mouse anti-LRRTM4 (clone N205B/22, UCDavis/NIH Neuromab, CA, USA), sheep anti-LRRTM4 (R&D Systems), rabbit anti-GPC4 (aa 88-101; Immundiagnostik), rabbit anti-GluR1 (Calbiochem), rabbit anti-synapsin (Millipore), chicken anti-MAP2 (Sigma), mouse anti-myc 9E10 (Santa Cruz Biotechnology), mouse anti-HA (Covance), mouse anti-FLAG M2 (Sigma), mouse anti-heparan sulfate delta (3G10 epitope) (USBiological).

Techniques: MANN-WHITNEY, Expressing