Journal: Bio-protocol

Article Title: Time-Lapse Super-Resolution Imaging and Optical Manipulation of Growth Cones in Elongating Axons and Migrating Neurons

doi: 10.21769/BioProtoc.5251

Figure Lengend Snippet: (A) Experimental scheme. CSPG-rich Matrigel corresponds to 60% Matrigel/Ncan/L15 medium. (B–D) Representative super-resolution time-lapse images of ventricular–subventricular zone (V-SVZ)-derived cultured migrating neurons expressing Venus-CAAX (green) and DsRed (red), which label membranes and cytosol, respectively. While “minute-interval” imaging reveals the overall dynamics of the leading process growth cone (B), “second-interval” imaging visualizes the dynamics of fine cellular structures such as lamellipodia and filopodia. (C, D) Yellow, light-blue, and magenta arrows indicate formed, buried, and retracted filopodium, respectively. The structure and dynamics of lamellipodial and filopodial structures should be additionally analyzed by using actin probes such as GFP-actin or EGFP-UtrCH. Numbers indicate seconds from the first imaging frame (B–D). Scale bars, 5 μm (B–D). CSPG, chondroitin sulfate proteoglycan.

Article Snippet: Recombinant Neurocan (Ncan) (R&D Systems, catalog number: 5800-NC-050) 28.

Techniques: Derivative Assay, Cell Culture, Expressing, Imaging

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: 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: Perineuronal net protein levels are altered in HIV-infected human brain tissue. Aggrecan and brevican were detected by Western blot using post-mortem human cortical lysates. Representative bands from infected and non-infected shown. A. Levels of the aggrecan (Acan) cleavage fragment (27 kDa) were elevated in HIV-infected samples (unpaired t test, df = 36, t = 2.086, p = 0.0441). B. Levels of intact full-length brevican (Bcan) (160 kDa) were decreased in HIV-infected samples (unpaired t test, df = 36, t = 5.985, p < 0.0001). C. Correlations between MMPs and PNN protein expression were assessed. MMP-3 levels were positively correlated with levels of aggrecan cleavage fragments (Pearson correlation; r = 0.4023, R2 = 0.1618, p = 0.0150, n = 36), and D. negatively correlated with full-length brevican (Pearson correlation; r = −0.4450, R2 = 0.1980, p = 0.0065, n = 36). E. There was a significant positive correlation between levels of MMP-13 and the aggrecan cleavage fragment (Pearson correlation; r = 0.9301, r2 = 0.8651, p < 0.0001, n = 36), and F. there was a trending negative correlation between levels of MMP-13 and brevican (Pearson correlation; r = −0.2928, r2 = 0.08575, p = 0.0831, n = 36). HIV-negative (black arrows) n = 15, HIV-positive (purple circles) n = 21; for all correlations the best fit line and 95% confidence bands were generated using linear regression. * p < 0.05, **** p < 0.0001.

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: Infection, Western Blot, Expressing, Generated

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, Clinical Proteomics, Membrane, Biomarker Discovery, 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, Control, Western Blot, Immunostaining, Enzyme-linked Immunosorbent Assay, Protein Binding, Positive Control, Recombinant, Incubation, Saline, 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: Control, Western Blot, Recombinant