recombinant bdnfpro  (Alomone Labs)

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: astrocytic BDNFpro secretion rescues LTP deficit in p75-flox mice. a Schematic representation of the experimental design. Step I, deletion of p75 NTR in astrocytes from tamoxifen-treated p75-flox mice precludes proBDNF transfer from neurons to astrocyte following TBS. Step II, LV-BDNFpro stop transduction replaces BDNFpro in astrocytes. Schematic representation of the experimental paradigms (right); mice were treated with tamoxifen (−5 to 0), injected with lentiviruses the last day of tamoxifen treatment (0 dptm) and finally recorded (14 dptm). LTP evoked in slices from p75-flox mice and control littermates injected with LV-GFP stop or LV-BDNFpro stop is shown. *** p

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFproVal/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNFCR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Mouse Assay, Transduction, Injection

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: BDNFpro restores memory retention in p75-flox mice. a Schematic diagram depicting the behavioral paradigm used for ORT. Mice were subjected to familiarization (sample phase) with two identical objects (circles). A test phase in which one familiar object (circle) is substituted with a novel one was performed after 10 min (square) and 24 h (triangle). b Schematic diagram depicting the experimental paradigm. p75-flox mice and control littermates treated with tamoxifen (−5 to 0) and injected with LV-GFP stop or LV-BDNFpro stop the last day of tamoxifen treatment (0 dptm) were subjected to ORT (14 dptm). Discrimination index is plotted against time interval between sample phase and test phases. ** p

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFproVal/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNFCR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Mouse Assay, Injection

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: post-synaptic targeting of TrkB/SorCS2 complex. a Schematic representation of the experimental design. Circular DNA probes (−) and (+) are coupled to II° antibody targeting αSorCS2 and αTrkB I° antibody. BDNFpro induces TrkB/SorCS2 complex formation (PLA TrkB/SorCS2 ) that is prevented in the presence of αSorCS2 (blocking) antibody. b Panels show PLA TrkB/SorCS2 signals in primary culture of cortical neurons treated with vehicle or BDNFpro. The insets show reference GFP-neurons. Scale bars: 5 μm. c Panels show a GFP-neuron treated with BDNFpro. Scale bar: 5 μm. Magnification of regions of interest 1 and 2 shows dendritic PLA TrkB/SorCS2 localization (red arrowheads). Scale bar: 1 μm. d Quantification of PLA TrkB/SorCS2 signal in cultured neurons treated with vehicle, BDNFpro (in presence or absence of αSorCS2), mBDNF or proBDNF CR . Data are presented as mean ± SEM; ** p

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFproVal/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNFCR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Proximity Ligation Assay, Blocking Assay, Cell Culture

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: BDNFpro-induced TrkB/SorCS2 targeting. a Schematic representation of the experimental design. Step I, deletion of p75 NTR in astrocytes from tamoxifen-treated p75-flox mice precludes proBDNF transfer from neurons to astrocyte following TBS. Step II, LV-BDNFpro stop transduction replaces BDNFpro in astrocytes. Step III, astrocytic BDNFpro provides final increase of TrkB/SorCS2 complexes in dendritic spines and LTP maintenance. b z-stack reconstruction showing NeuN/PLA TrkB/SorCS2 colocalization signal in TBS-slices from p75-flox mice transduced with LV-GFP stop or LV-BDNFpro stop . Scale bars: 40 μm. The insets show the field of analysis. Scale bars: 15 μm. NeuN/PLA TrkB/SorCS2 colocalization was quantified using Mander’s overlap. ** p

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFproVal/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNFCR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Mouse Assay, Transduction, Proximity Ligation Assay

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: BDNFpro expression in cortical astrocytes. a Schematic representation of proBDNF precursor and cleaved BDNFpro domain. αBDNFpro antibody recognizes the furin cleavage site of the prodomain. Western blotting probing recombinant mBDNF, BDNFpro, and proBDNF CR with αBDNFpro and αmBDNF antibodies. b Cortical slices from control mice injected with AAV-GFAP-GFP virus were recorded and fixed 10 min after TBS for immunostaining. z-stack reconstruction shows astrocytes labeled by GFP. Magnification of a single stack from a region of interest (ROI) shows BDNFpro immunoreactivity and BDNFpro/GFP colocalization signal of one GFP-astrocyte delimited by an approximate territory (white dashed). Scale bars: 10 µm. c z-stack reconstruction of BDNFpro/GFP colocalization signals in astrocytes from baseline- and TBS-slices from control mice. The insets show GFP signal. BDNFpro/GFP colocalization was quantified in the whole cell and branches using Mander’s overlap. *** p

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFproVal/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNFCR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Expressing, Western Blot, Recombinant, Mouse Assay, Injection, Immunostaining, Labeling

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: subcellular localization of BDNFpro. a Graphical representation of the SIM super-resolution microscope. 3D-SIM image of a GFP-labeled astrocyte in a TBS-slice from control mice. Scale bar: 10 µm. Magnification of a ROI shows BDNFpro/GFP colocalization signal localized in fine membrane extensions of the cell periphery. Scale bar: 200 nm. b 3D-SIM image of the ROI in ( a ); z-axe is visualized in pseudocolor to facilitate microdomains identification. Scale bar: 200 nm. Magnification of microdomains characterized by the typical fingerlike extension (dashed squares 1 and 2) and flat lamellar sheath (dashed squares 3 and 4) are shown. BDNFpro/GFP colocalization is indicated (red arrowheads). Scale bars: 40 nm.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFproVal/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNFCR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Microscopy, Labeling, Mouse Assay

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: vesicular localization of BDNFpro. a z-stack reconstruction shows astrocytes labeled by GFP. Cortical slices from control mice injected with AAV-GFAP-GFP virus were fixed 10 min after TBS and processed for immunostaining and confocal analysis. Scale bar: 10 µm. Magnification of a ROI shows one GFP-astrocyte delimited by an approximate territory (white dashed). Scale bar: 10 µm. BDNFpro/GFP and Vamp2/GFP co-localizations signals are shown. Magnification shows representative areas (dashed squares 1 to 4) in which BDNFpro/GFP and Vamp2/GFP signals overlap. Scale bars: 1 µm. b 3D-SIM image of a GFP-labeled astrocyte in a TBS-slice from control mice. Scale bar: 10 µm. Magnification of a ROI shows BDNFpro/Vamp2 colocalization signal. Scale bar: 500 nm. Magnification shows BDNFpro/Vamp2 colocalization signal in fine membrane extensions of the cell periphery (dashed squares 1 to 4). Scale bars: 50 nm. c EM image depicts BDNFpro-gold at astrocytic microdomains (light blue) surrounding an axon bouton. Scale bar: 100 nm. Magnification of the ROI shows gold particles (red arrowheads) in vesicular-like structures. Scale bar: 20 nm. d Digital reconstruction of the image in ( c ). Astrocytic vesicles (black boundary) are shown.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFproVal/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNFCR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Labeling, Mouse Assay, Injection, Immunostaining

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: localization of BDNFpro in astrocytic microdomains. a Experimental design linking field-potential with electron microscopy (EM) in layer II/III perirhinal cortex. TBS (10 min)-slices were dissected for EM processing. b Representative EM-image depicts BDNFpro-gold particles at axon bouton (dashed squares 1 to 4) and dendritic spine (dashed squares 5 and 6). Scale bar: 100 nm. Magnification indicates representative areas (dashed squares 1 to 6) in which gold particles (red arrowheads) localization is shown. Scale bars: 10 nm. c Representative EM-image depicts BDNFpro-gold particles (dashed squares 1 to 6) at astrocytic microdomains (light blue) Scale bar: 250 nm. Magnification indicates representative areas (dashed squares 1 to 6) in which gold particles (red arrowheads) localization is shown. Scale bars: 20 nm. d Dot plot depicts the number of BDNFpro-gold particles in whole astrocytes and peri-synaptic astrocytes counted per section ( n = 41 sections, 5 slices, 3 mice). e Dot plot depicts the percentage of BDNFpro-gold particles at peri-synaptic astrocytes ( n = 41 sections, 5 slices, 3 mice). Data are mean ± SEM.

Article Snippet: In some experiments, recombinant BDNFpro (10 ng/ml; Alomone Labs, Cat#B-245), BDNFproVal/Met (10 ng/ml; Alomone Labs, Cat#B-445), mBDNF (10 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy), and proBDNFCR (20 ng/ml; Laboratory of Antonino Cattaneo, SNS, Pisa, Italy) were perfused into a recording chamber.

Techniques: Electron Microscopy, Mouse Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

doi: 10.1074/jbc.M109.062364

Figure Lengend Snippet: Differential binding of pro-BDNF and neurotension to sortilin and pro-sortilin. A , SPR analysis showing that unprocessed pro-BDNF (50 n m ) binds nearly as efficiently to the receptor in the presence (pro-sortilin) as in the absence (sortilin) of the receptor

Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

Techniques: Binding Assay, SPR Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

doi: 10.1074/jbc.M109.062364

Figure Lengend Snippet: Selective competition of ligands by sortilin-derived peptide antagonist. SPR binding analysis of 50 n m unprocessed pro-BDNF ( A ), 50 n m unprocessed pro-NGF ( B ), and 90 n m RAP ( C ) to immobilized sortilin in the absence and presence of the sort166–181

Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

Techniques: Derivative Assay, SPR Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

doi: 10.1074/jbc.M109.062364

Figure Lengend Snippet: Mutation of the linear binding site specifically impairs binding of both the NGF and the BDNF pro-domains. SPR analysis showing reduced binding of equal amounts (analyte concentration: 200 n m ) of the soluble extracellular domains of sortilin-4A compared

Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

Techniques: Mutagenesis, Binding Assay, SPR Assay, Concentration Assay