recombinant mouse neurocan  (R&D Systems)

Journal: Experimental neurology

Article Title: Increased matrix metalloproteinase levels and perineuronal net proteolysis in the HIV-infected brain; relevance to altered neuronal population dynamics

doi: 10.1016/j.expneurol.2019.113077

Figure Lengend Snippet: MMPs cleave the perineuronal net proteins aggrecan and brevican. Active recombinant matrix metalloproteinases were incubated with recombinant aggrecan and brevican. In vitro digests revealed that A. aggrecan (Acan) and B. brevican (Bcan) are both cleaved by MMP-3 and MMP-13, as demonstrated by the appearance of the indicated cleavage fragments. Enzymatic cleavage is prevented by addition of the broad-spectrum MMP inhibitor GM6001.

Article Snippet: Recombinant human aggrecan (R&D, catalog # 1220-PG-025) was used at a concentration of 62.5 μg/mL and recombinant human brevican (R&D, catalog # 5800-NC-050) was used at a concentration of 50 μg/mL.

Techniques: Recombinant, Incubation, In Vitro

Journal: Frontiers in Cellular Neuroscience

Article Title: Neurocan Inhibits Semaphorin 3F Induced Dendritic Spine Remodeling Through NrCAM in Cortical Neurons

doi: 10.3389/fncel.2018.00346

Figure Lengend Snippet: Localization of Neurocan in mouse medial frontal cortex (MFC) by immunogold labeling and electron microscopy. (A) Electron micrograph of MFC layer 2/3 at P18, showing immunogold labeling of Neurocan near the plasma membrane adjacent to a spine (Sp) and axon terminal (AT; arrows). (B) Neurocan labeling in the extracellular space near an axon terminal (AT; arrow) at P18 [Nucleus (Nuc) and cytoplasm (Cyto)]. (C) Accumulation of Neurocan (arrows) in extracellular space and along the plasma membrane of a dendrite (D) at P18. Mitochondria (M) were unlabeled. (D) Neurocan labeling adjacent to axon terminals (AT) at P18. (E) Neurocan labeling at neck of spine (Sp) and near excitatory synapses (arrows) at P80. Scale bar = 1 μm. (F) Validation of Neurocan antibody specificity by immunoperoxidase staining of COS-7 cells transfected with Neurocan-AP or AP alone in the APtag5 vector, using Neurocan antibodies or no primary antibody. An antibody dilution series was carried out in pilot experiments. Scale bar = 50 μm.

Article Snippet: As described in , transfected cells at DIV14 were treated with 3 nM Sema3F-Fc (R&D) or Fc (Abcam) for 30 min. Where indicated, cultures were pre-treated for 30 min with 8–20 nM full length recombinant human Neurocan (Glu23-Cys1321, R&D) or a mouse Neurocan fragment (Asp23-Asp637, R&D), which lacks the C-terminal sushi domain and approximately half of the GAG-modified region.

Techniques: Labeling, Electron Microscopy, Immunoperoxidase Staining, Transfection, Plasmid Preparation

Journal: Frontiers in Cellular Neuroscience

Article Title: Neurocan Inhibits Semaphorin 3F Induced Dendritic Spine Remodeling Through NrCAM in Cortical Neurons

doi: 10.3389/fncel.2018.00346

Figure Lengend Snippet: Cell binding and Neurocan interaction with NrCAM. (A) COS-7 cells transfected with vector alone (pCAGGS-IRES-mEGFP) or pCAGGS-NrCAM-IRES-mEGFP were pre-treated with 8 nM Neurocan, then fixed and subjected to immunofluorescence staining without permeabilization to detect surface-bound Neurocan (red). Scale bar = 100 μm. (B) Mean fluorescence intensity (±SEM) of Neurocan immunofluorescence staining on the surface of COS-7 cells, as shown in panel A . NrCAM-expressing cells treated with Neurocan showed significantly greater levels of bound Neurocan than untreated cells. Fluorescence intensity in cells with vector alone treated with Fc or Sema3F-Fc was non-significant (ns). ∗ p > 0.05, t -test, n = 5 images each condition. (C) Lysates (50 μg) of cells transfected with vector alone or pCAGGS-NrCAM-IRES-mEGFP were treated with Neurocan as in panel A , and immunoblotted (IB) with Neurocan antibodies. Blots were reprobed with antibodies directed against GAPDH (loading control) or NrCAM (expression control). Representative immunoblots of three experiments are shown. (D) Mouse cortical neuron cultures from NrCAM null mice were transfected with vector alone or pCAGGS-NrCAM-IRES-EGFP, and pre-treated with 20 nM Neurocan before fixation and immunostaining to detect surface-bound Neurocan. In merged images of EGFP (green) and Neurocan (red), more Neurocan immunofluorescence was observed on the surface of neurons expressing NrCAM than on NrCAM null neurons with vector alone. Scale bar = 50 μm. (E) Mean fluorescence intensity (±SEM) of surface-bound Neurocan immunostaining on neurons in panel D NrCAM-expressing cells treated with Neurocan showed significantly greater levels of bound Neurocan than NrCAM-minus neurons. ∗ p > 0.05, t -test, and n = 10 neurons per condition. (F) ELISA of Neurocan-AP or control AP protein binding to NrCAM-Fc or positive control NCAM-Fc on protein A-coated microtiter wells. AP binding was detected colorimetrically with p-nitrophenylphosphate. The mean (±SEM) optical densities (OD 405) of Neurocan-AP bound to NrCAM-Fc or NCAM-Fc were significantly greater than control AP ( t -test and ∗ p > 0.05). (G) Recombinant human Neurocan was incubated in Tris buffered saline with purified Fc, Sema3F-Fc, or Sema3A-Fc proteins, then complexes were pulled down with Protein A/G Sepharose beads. Immunoblotting for Neurocan showed no binding of Neurocan to Fc or Sema3F-Fc, whereas Neurocan bound effectively to Sema3A-Fc. Blots were reprobed with anti-Fc antibodies to demonstrate that equivalent amounts of Fc fusion proteins were pulled down. Recombinant Neurocan (left lane) ran as a broad band between 250 and 130 kDa.

Article Snippet: As described in , transfected cells at DIV14 were treated with 3 nM Sema3F-Fc (R&D) or Fc (Abcam) for 30 min. Where indicated, cultures were pre-treated for 30 min with 8–20 nM full length recombinant human Neurocan (Glu23-Cys1321, R&D) or a mouse Neurocan fragment (Asp23-Asp637, R&D), which lacks the C-terminal sushi domain and approximately half of the GAG-modified region.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Fluorescence, Expressing, Western Blot, Immunostaining, Enzyme-linked Immunosorbent Assay, Protein Binding, Positive Control, Recombinant, Incubation, Purification

Journal: Frontiers in Cellular Neuroscience

Article Title: Neurocan Inhibits Semaphorin 3F Induced Dendritic Spine Remodeling Through NrCAM in Cortical Neurons

doi: 10.3389/fncel.2018.00346

Figure Lengend Snippet: Enzymatic digestion of Neurocan GAG chains with chondroitinase ABC decreases its ability to inhibit Sema3F-induced spine retraction. (A) Images showing spines on apical dendrites from cortical neurons (EGFP, green) in culture treated with Fc or Sema3F-Fc. Neurocan blocked Sema3F-mediated spine retraction, whereas chABC-treated Neurocan was not effective. Scale bar = 10 μm. (B) Quantification of experiment in panel A shows a significant reduction in mean spine density of control neurons treated with Sema3F-Fc compared to Fc. Sema3F-induced spine retraction was fully blocked by 20 nM Neurocan, as well as by chABC-digested Neurocan ( ∗ p < 0.05, t -test; n = 3, 10 neurons per condition). (C) Immunoblotting of Neurocan before and after treatment with chABC to remove GAG chains. A shift in apparent molecular size of chABC-treated Neurocan was observed, reflecting a decrease in GAG content. (D) Mouse cortical neurons with and without pre-treatment with recombinant mutNeurocan lacking the C-terminal sushi domain (20 nM, 30 min) showed the mouse Neurocan fragment inhibited Sema3F-Fc induced spine retraction. (E) Model showing that interaction of the PNN protein Neurocan with NrCAM on the surface of dendritic spines in cortical pyramidal neurons terminates Sema3F-induced dendritic spine remodeling during postnatal maturation. Neurocan core protein is depicted in green with yellow GAG chains. The Sema3F receptor complex is composed of NrCAM (yellow), Npn2 (blue), and PlexA3 (red) subunits.

Article Snippet: As described in , transfected cells at DIV14 were treated with 3 nM Sema3F-Fc (R&D) or Fc (Abcam) for 30 min. Where indicated, cultures were pre-treated for 30 min with 8–20 nM full length recombinant human Neurocan (Glu23-Cys1321, R&D) or a mouse Neurocan fragment (Asp23-Asp637, R&D), which lacks the C-terminal sushi domain and approximately half of the GAG-modified region.

Techniques: Western Blot, Recombinant

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: GAG-modified neurocan blocks chABC-induced decrease of perisomatic synaptic puncta in organotypic brain slices. ( A ) Immunostaining of neuronal soma (NeuN), a parvalbumin-positive interneuron (tdTomato), and a perineuronal net (WFA) in DIV14 organotypic brain slice culture. Scale bar = 10 μm. ( B ) WFA labeling of perineuronal nets in control penicillinase and chABC-treated brain slices. Scale bar = 30 μm. ( C ) Representative image of perisomatic synapses (tdTomato) in control penicillinase or chABC-treated slice cultures. Representative perisomatic puncta around a single soma are indicated with arrowheads. Scale bar = 10 μm. ( D ) Quantification of the mean number of perisomatic synaptic puncta per soma (n = 30 soma/condition, 3 animals per condition, t-test, *p < 0.05). ( E ) NCAM was immunoprecipitated from brain lysates, followed by immunoblotting with antibodies against neurocan, versican, or aggrecan/brevican (using an antibody raised against shared epitope). ( F ) Slices were treated with control penicillinase or chABC as in ( C ) followed by rescue with neurocan or tenascin-R. Quantification of the mean number of perisomatic synapses per soma was performed (>90 soma per mouse per condition, n = 3 mice, two-way ANOVA with Bonferonni post-hoc testing, *p < 0.05). ( G ) Immunoblot to detect recombinant proteins (immunoblotted for His tag) and HNK-1 carbohydrate modification. P21 brain lysate was used as a positive control for HNK-1 signal.

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Modification, Immunostaining, Slice Preparation, Labeling, Control, Immunoprecipitation, Western Blot, Recombinant, Positive Control

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan binds the Ig2 domain of NCAM, decreasing EphA3 binding. ( A ) Fc-pulldowns of the NCAM extracellular domain (NCAM-EC), truncation mutants of NCAM, or control Fc and recombinant neurocan. ( B ) Densitometry of ( A ) indicating the level of neurocan bound (relative to positive control NCAM-EC-Fc bound neurocan) for each construct (*p < 0.05 compared to NCAM-EC-Fc) (C) Fc-pulldowns of NCAM-EC-Fc or control Fc with mouse neurocan (lacking sushi domain) and full-length human neurocan. ( D ) Co-immunoprecipitation of WT NCAM-140 or mutants of NCAM and neurocan from transfected HEK293T cells. ( E ) Densitometry of ( D ). The amount of co-immunoprecipitated neurocan for each NCAM IP was normalized to control WT NCAM-bound neurocan (n = 3, t-test, *p < 0.05). ( F ) Immunoblot of untreated or chABC-treated neurocan protein probed for neurocan or C-4-S. ( G ) Co-immunoprecipitation of NCAM-140 and EphA3 from transfected HEK293 cells treated with no neurocan (control), neurocan, or chABC-treated neurocan. ( H ) Densitometry of ( G ). The amount of co-immunoprecipitated EphA3 for each NCAM IP was normalized to control NCAM-bound EphA3 (n = 3, t-test, *p < 0.05).

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Binding Assay, Control, Recombinant, Positive Control, Construct, Immunoprecipitation, Transfection, Western Blot

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan impairs ephrin-A5-mediated clustering of NCAM and EphA3 in cortical interneurons in culture. ( A ) Cortical neuron cultures were pretreated with no neurocan (control) or neurocan followed by preclustered Fc or ephrin-A5-Fc, and localization of endogenous NCAM (green) and EphA3 (red) was assessed in axons of GABA immunopositive axons by confocal microscopy. Scale bars = 5 μM. ( B ) Pearson’s Correlation Coefficients (R-Total) were generated for each condition using ImageJ co-localization software (n = 3, two-way ANOVA with Bonferonni post-hoc testing, *p < 0.05).

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Control, Confocal Microscopy, Generated, Software

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan decreases ephrin-A5-induced EphA3 autophosphorylation. ( A ) HEK293T cells transfected with NCAM and EphA3 were treated with preclustered control Fc or ephrin-A5-Fc, and EphA3 was immunoprecipitated. EphA3 autophosphorylation was assessed by immunoblotting with a phosphotyrosine antibody (PY99). Total levels of immunoprecipitated EphA3 were assessed by reprobing with EphA3 antibody. ( B ) Densitometry of ( A ). Graph indicates the ratio of phosphotyrosine to EphA3 values for each condition (n = 3, *p < 0.05). ( C ) Fc-pulldowns of control Fc, NCAM-EC-Fc, and ephrin-A5-Fc with recombinant neurocan. Level of neurocan bound (relative to positive control NCAM-EC-Fc bound neurocan) is indicated as a percentage under each lane. ( D ) Co-immunoprecipitation of NCAM (positive control) or EphA3 with neurocan from transfected HEK293T cells. Level of neurocan bound (relative to positive control NCAM-EC-Fc bound neurocan) is indicated as a percentage under each lane.

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Transfection, Control, Immunoprecipitation, Western Blot, Recombinant, Positive Control

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan inhibits ephrin-A5-induced growth cone collapse in GABAergic interneurons. ( A ) Representative spread and collapsed growth cones of GABA-immunostained interneurons in cortical neuron cultures. Scale bar = 5 μM. ( B ) The percentage of collapsed growth cones was determined for each condition (300 growth cones per condition, n = 3 experiments, 2-way ANOVA, Bonferonni post-hoc testing, ***p < 0.001).

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques:

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Model of inhibition of NCAM/EphA3 clustering and activation by neurocan. During postnatal remodeling of transient perisomatic synapses made by PV + interneurons onto pyramidal cell soma, ephrin-A5 dimers bind the ligand binding domains (LBD) of an EphA3 dimer. NCAM clusters the EphA3 receptors through binding of the NCAM Ig2 domain to the EphA3 CRD. EphA3 clustering activates tyrosine kinase signaling leading to synapse retraction. With further maturation, neurocan in PNNs engages the Ig2 domain of non-PSA NCAM, inhibiting EphA3 clustering and retraction of inhibitory perisomatic contacts. NCAM is shown in black, EphA3 is green, and ephrin-A5 is yellow. Neurocan core protein is depicted in purple with GAG chains in orange on a PNN scaffold (blue). P = phosphorylation. The illustration was created by modifying images purchased in the PPT Drawing Toolkits-BIOLOGY Bundle from Motifolio, Inc.

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Inhibition, Activation Assay, Ligand Binding Assay, Binding Assay, Phospho-proteomics