Journal: Molecular Neurodegeneration

Article Title: Axotomy-induced neurotrophic withdrawal causes the loss of phenotypic differentiation and downregulation of NGF signalling, but not death of septal cholinergic neurons

doi: 10.1186/1750-1326-5-5

Figure Lengend Snippet: Effect of septo-hippocampal pathway axotomy in cholinergic septal neurons . A , Light microscopy of coronal sections stained for AChE 3 or 21 days after lesion procedure, showing the early effect of axotomy in cholinergic projections towards the hippocampus. Twenty-one days after axotomy, AChE-positive terminals are strongly reduced in the ipsilateral side of the lesion, as compared to the contralateral side (scale bar 1 mm). Inferior panels, magnification inset of AChE-positive neurites in dentate gyrus (scale bar 200 μm). B , Light microscopy of immunohistochemistry anti-ChAT in coronal sections, showing the effects of the axotomy in cell bodies of cholinergic septal neurons, 3 or 21 days after axotomy. Twenty-one days after the lesion, the number of ChAT-positive neurons is clearly reduced on the ipsilateral side of the medial septum (CL, contralateral; IL, ipsilateral. Scale bar: 1 mm). C , Time course of ChAT- and p75-positive neuron loss after axotomy. The graph shows the quantification of ChAT- or p75-immunopositive neurons in the medial septum from serial sections of brains at 3, 7, 14 and 21 days after the lesion. Note that the numbers of ChAT- and p75-immunopositive cells decay with similar kinetics but with a different slope between 7-14 days.

Article Snippet: Rabbit anti-NGF (Alomone Labs, Jerusalem, Israel) and rabbit anti-p75 (Upstate, NY, USA) were used at 1:300 and 1:500, respectively.

Techniques: Light Microscopy, Staining, Immunohistochemistry

Journal: Molecular Neurodegeneration

Article Title: Axotomy-induced neurotrophic withdrawal causes the loss of phenotypic differentiation and downregulation of NGF signalling, but not death of septal cholinergic neurons

doi: 10.1186/1750-1326-5-5

Figure Lengend Snippet: The number of neurons that are positive only for p75 is increased in the ipsilateral side after axotomy . A , Confocal microscopy of double-immunofluorescence anti-ChAT/anti-p75 reveals an increase in the number of neurons that are positive only for p75 at different times after the axotomy. The superior panel is a panoramic view of the medial septum at 14 days after axotomy (scale bar: 1 mm). The central and inferior panels are magnification insets (scale bar: 200 μm) of neurons contralateral and ipsilateral to the lesioned side, showing p75-positive and ChAT-negative cells as indicated by the white arrows. B , The graph shows the percentage of total septal neurons expressing p75 that are p75-positive and ChAT-negative at 1, 3, 7 or 14 days after axotomy. Black bars represent the control side of the septum and white bars represent the side ipsilateral to the lesion. Asterisk indicates significance level p < 0.05.

Article Snippet: Rabbit anti-NGF (Alomone Labs, Jerusalem, Israel) and rabbit anti-p75 (Upstate, NY, USA) were used at 1:300 and 1:500, respectively.

Techniques: Confocal Microscopy, Immunofluorescence, Expressing

Journal: Molecular Neurodegeneration

Article Title: Axotomy-induced neurotrophic withdrawal causes the loss of phenotypic differentiation and downregulation of NGF signalling, but not death of septal cholinergic neurons

doi: 10.1186/1750-1326-5-5

Figure Lengend Snippet: p75-positive and ChAT-negative neurons are not GABAergic . Confocal microscopy of triple-immunofluorescence anti-ChAT/anti-p75/anti-parvalbumin shows no significant colocalization of GABAergic marker and p75. Left, a panoramic view of the medial septum (scale bar: 1 mm) and panel showing a magnification inset of a representative group of neurons from triple-labeled sections (scale bar: 300 μm).

Article Snippet: Rabbit anti-NGF (Alomone Labs, Jerusalem, Israel) and rabbit anti-p75 (Upstate, NY, USA) were used at 1:300 and 1:500, respectively.

Techniques: Confocal Microscopy, Immunofluorescence, Marker, Labeling

Journal: Molecular Neurodegeneration

Article Title: Axotomy-induced neurotrophic withdrawal causes the loss of phenotypic differentiation and downregulation of NGF signalling, but not death of septal cholinergic neurons

doi: 10.1186/1750-1326-5-5

Figure Lengend Snippet: Cholinergic septal neurons do not undergo apoptosis after axotomy . A , Triple-immunofluorescence against Neu-N (neuronal marker), GFAP (astroglial marker) and activated Caspase-3 shows no colocalization of the apoptotic marker with neurons at different time points (1, 3, 7, 14 days) after axotomy. There is an increase in the number of cells that are immunopositive for activated caspase-3 with time. The correlation between GFAP and activated Caspase-3 suggests that astrocytes are undergoing apoptosis (scale bar: 50 μm). B , Triple-immunofluorescence against ChAT, p75 and p53, shows no colocalization of p53 (an early apoptotic marker) with p75- or ChAT-immunopositive neurons in the brains of axotomized rats 14 days after the axotomy. Confocal microscopy (scale bar: 50 μm). Triple-immunofluorescence against ChAT, p75 and activated caspase-3 shows no colocalization of activated Caspase-3 and p75 or ChAT immunopositive neurons after 14 days of axotomy. Confocal microscopy (scale bar 50 μm). C , Axotomized septal neurons are not labeled with Fluorojade C (a specific staining for degenerating neurons). Superior panel, a brain section from a rat injected in medial septum with 100 mM H 2 O 2 was stained with Neurotrace and Fluorojade C as a positive control. The arrow indicates a degenerating neuron. Center panel, double-labeling with Neurotrace and Fluorojade in a brain section from an untreated rat. Inferior panel shows no colocalization of Neurotrace and Fluorojade C in medial septal neurons 14 days after axotomy (scale bar 50 μm).

Article Snippet: Rabbit anti-NGF (Alomone Labs, Jerusalem, Israel) and rabbit anti-p75 (Upstate, NY, USA) were used at 1:300 and 1:500, respectively.

Techniques: Immunofluorescence, Marker, Confocal Microscopy, Labeling, Staining, Injection, Positive Control

Journal: Molecular Neurodegeneration

Article Title: Axotomy-induced neurotrophic withdrawal causes the loss of phenotypic differentiation and downregulation of NGF signalling, but not death of septal cholinergic neurons

doi: 10.1186/1750-1326-5-5

Figure Lengend Snippet: There is no difference between the number of septal neurons when comparing the ipsilateral and contralateral regions of brains 21 days after axotomy . A , Left, confocal microscopy of triple labeling for Neurotrace (red), NeuN (green) and GFAP (blue) from the septal region of a lesioned brain. In order to assure that we were counting only neurons, we used two neuronal markers and an astrocytic marker (GFAP). Right, inset magnification of a neuron (scale bar: 80 μm) showing the Neurotrace and NeuN labeling profiles. B , Diagram illustrating the area of the medial septum (MS) that was considered for the quantification of cholinergic or total number of neurons (adapted from Rat Brain Atlas ). Each side of the total area was divided to 4 fields and then photographed and manually quantified. The anatomical landmarks used to define the MS are also indicated (see Experimental Methods). cc, corpus callosum; LV, lateral ventricle; aca, anterior commissure. C , Quantification of p75-positive, ChAT-positive and total septal neurons. Comparison of the number of septal neurons on contralateral and ipsilateral sides shows differences in the numbers of p75- and ChAT-immunopositive neurons, but no significant difference in total number of neurons (n = 5; Student's t-test, p > 0.001, ± SD) 21 days after axotomy.

Article Snippet: Rabbit anti-NGF (Alomone Labs, Jerusalem, Israel) and rabbit anti-p75 (Upstate, NY, USA) were used at 1:300 and 1:500, respectively.

Techniques: Confocal Microscopy, Labeling, Marker

Journal: Molecular Vision

Article Title: Electroporation-mediated gene delivery of cleavage-resistant pro–nerve growth factor causes retinal neuro- and vascular degeneration

doi:

Figure Lengend Snippet: Overexpression of proNGF reduced NGF and induced expression of TrkA and p75 NTR , but not sortilin. A , B : Western blot (WB) analysis of rat retinal lysate showed significant (1.5-fold) increase in expression of TrkA and p75 NTR in rats electroporated with pGFP-proNGF123 as compared with those electroporated with pGFP (n=4). C : WB analysis showed that overexpression of pGFP-proNGF123 in rat retinas did not affect sortilin protein expression level as compared with the control rats (n=4). D : WB analysis showed that overexpression of pGFP-proNGF123 significantly reduced NGF expression (n=5) compared with GFP controls. The asterisk represents significant difference as compared with control group at p<0.05. ROD, relative optical density.

Article Snippet: Antibodies for proNGF and NGF (Alomone Labs Ltd), p75 NTR , sortilin, and TrkA (Millipore) were used.

Techniques: Over Expression, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

doi: 10.3390/ijms23073849

Figure Lengend Snippet: Induction of p75NTR expression in human neuroblastoma cell lines by VPA and entinostat. ( A ) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y and LAN-1 cells were treated as indicated in ( A ) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. ( C ) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.

Article Snippet: For analysis of p75NTR expression, nonpermeabilized cells were blocked with 3% BSA and incubated overnight with rabbit polyclonal ATTO-488-conjugated anti-p75NTR (extracellular) antibody (cat. no. ANT-007-AG, Alomone Labs, Jerusalem, Israel) (1:100).

Techniques: Expressing, Western Blot, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

doi: 10.3390/ijms23073849

Figure Lengend Snippet: Enhancement of cell surface p75NTR expression by VPA and entinostat. ( A ) The SH-SY5Y cells were incubated for 24 h with either vehicle, 1 mM VPA, or 1 µM entinostat. Thereafter, the cells were treated with the cell impermeant biotinylating agent sulfosuccinimidyl-6-(biotin-amido) hexanoate and the solubilized proteins were isolated by precipitation with streptavidin-conjugated agarose beads. The total cell extract (cell lysate) and precipitated proteins (surface protein) were analyzed for p75NTR by Western blot. The bar graphs report the changes in the cell surface p75NTR levels that were normalized normalized to pan-cadherin (cadherin), a plasma membrane marker. Values are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t -test. ( B ) SH-SY5Y cells that were grown on glass coverslips were treated for 24 h with either vehicle ( a ) or 1 µM entinostat (entin) ( b , c ), fixed, and incubated overnight with ATTO-488-conjugated anti-p75NTR (extracellular) antibody. In ( c ) the cells were preincubated with a mouse monoclonal unconjugated antibody directed against the extracellular domain of the receptor before the addition of the labeled antibody. The images were analyzed for p75NTR expression (green color) by fluorescence microscopy. The nuclei were stained in blue with 4′-6-diamidino-2phenylindole dihydrochloride (DAPI). Scale bar = 25 µm. The values that are shown in the scatter plot are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t -test.

Article Snippet: For analysis of p75NTR expression, nonpermeabilized cells were blocked with 3% BSA and incubated overnight with rabbit polyclonal ATTO-488-conjugated anti-p75NTR (extracellular) antibody (cat. no. ANT-007-AG, Alomone Labs, Jerusalem, Israel) (1:100).

Techniques: Expressing, Incubation, Isolation, Western Blot, Marker, Labeling, Fluorescence, Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

doi: 10.3390/ijms23073849

Figure Lengend Snippet: Exposure to p75IgG-Sap induces p75NTR internalization, intracellular saporin-S6 delivery, and citotoxicity in entinostat-treated SH-SY5Y neuroblastoma cells. ( A ) The cells were treated for 24 h with 1 µM entinostat and the cell lysates were analyzed for p75NTR immunoreactivity by Western blot using either a non-targeted preimmune antibody conjugated to saporin-S6 (IgG-Sap) (1:50) or p75IgG-Sap (1:50). The images are representative of three separate experiments. ( B ). The cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat (entin) and then exposed for additional 24 h with either vehicle or p75IgG-Sap (30 nM). Biotinylated cell surface proteins were isolated and analyzed for p75NTR expression. The values are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle; ### , p < 0.001 by ANOVA followed by by Tukey’s test. ( C ) The cells were treated with 1 µM entinostat for 24 h and then exposed to either p75IgG-Sap (30 nM) or IgG-Sap (30 nM) for additional 24 h. The cells were then analyzed for p75NTR (green color) and LAMP1 (red color) localization by immunofluorescence. ( D ) The cells were incubated for 24 h with either vehicle or entinostat (1 µM), exposed to p75IgG-Sap (30 nM) for additional 24 h, and then analyzed for saporin-S6 (green color) and LAMP1 (red color) expression by immunofluorescence. In ( C , D ) the cell nuclei were stained in blue with DAPI. Scale bar = 25 µm. Values that are reported in the scatter plots are the mean ± SD of three independent experiments # p < 0.05; ## p < 0.01 by Student’s t -test. ( E ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and subsequently exposed to the indicated concentrations of p75IgG-Sap for 24 h. Cell viability was assayed by propidium iodide (PI) staining (red color). The values are the mean ± SD of five independent experiments. * p < 0.05 vs. vehicle; # p < 0.05, ### p < 0.001 by ANOVA followed by Tukey’s test. ( F ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and then exposed to either vehicle or 30 nM p75IgG-Sap. The images were obtained by phase-contrast microscopy and are representative of five separate experiments. ( G – J ) Cell viability was determined by a luminescence assay; ( G ) The cells were treated as indicated in ( F ); ( H , I ) The cells were treated with either vehicle or 1 µM entinostat and then incubated for 24 h with vehicle, 30 nM IgG-Sap, or 30 nM saporin-S6 (Sap); ( J ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and then incubated with 30 nM p75IgG-Sap in the absence and in the presence of 100 nM p75IgG added 2 h before the immunotoxin. The values are expressed as percent of control (vehicle) the mean ± SD of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle; ### p < 0.001, ns = not significant by ANOVA followed by Tukey’s test.

Article Snippet: For analysis of p75NTR expression, nonpermeabilized cells were blocked with 3% BSA and incubated overnight with rabbit polyclonal ATTO-488-conjugated anti-p75NTR (extracellular) antibody (cat. no. ANT-007-AG, Alomone Labs, Jerusalem, Israel) (1:100).

Techniques: Western Blot, Incubation, Isolation, Expressing, Immunofluorescence, Staining, Microscopy, Luminescence Assay

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

doi: 10.3390/ijms23073849

Figure Lengend Snippet: Upregulation of p75NTR and induction of apoptosis by entinostat in neuroblastoma spheroids. ( A , D ) Light microscopy images of IMR-32 and SH-SY5Y multicell spheroids (mcs) that were incubated for 72 h with either vehicle or 1 µM entinostat. The scatter plots report the values of mcs sizes expressed as percent of control (vehicle). Scale bar = 200 µm. ( B , C , E , F ) The spheroids were treated as indicated in ( A , D ) and then analyzed for p75NTR levels and PARP cleavage by Western blot. The values are the mean ± SD of four individual experiments. ** p < 0.01, *** p < 0.001 vs. vehicle by Student’s t -test.

Article Snippet: For analysis of p75NTR expression, nonpermeabilized cells were blocked with 3% BSA and incubated overnight with rabbit polyclonal ATTO-488-conjugated anti-p75NTR (extracellular) antibody (cat. no. ANT-007-AG, Alomone Labs, Jerusalem, Israel) (1:100).

Techniques: Light Microscopy, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

doi: 10.3390/ijms23073849

Figure Lengend Snippet: Expression of p75NTR and histone H3 acetylation in neuroblastoma tumor xenografts and different organs of entinostat-treated mice. ( A , C – F ) Athymic nude mice bearing xenograft of SH-SY5Y cells were treated daily for 10 days with either the vehicle or entinostat (20 mg/kg) by oral gavage. The mice were sacrificed, the tumor xenografts and the indicated organs were resected, and the tissue extracts were analyzed for p75NTR levels by Western blot. ( B ) The levels of acetylated histone H3 were determined in neuroblastoma tumor xenografts of vehicle- and entinostat-treated mice by Western blot. Each lane was loaded with a sample that was obtained from an individual animal (in ( A , B ): four animals that were treated with vehicle and five animals that were treated with entinostat; in ( C – F ), three animals that were treated with either vehicle or entinostat). Scatter plots indicate the absolute values of densitometric ratios and are the mean ± SD of three independent Western blots. * p < 0.05, *** p < 0.001, ns = not significant by Student’s t test.

Article Snippet: For analysis of p75NTR expression, nonpermeabilized cells were blocked with 3% BSA and incubated overnight with rabbit polyclonal ATTO-488-conjugated anti-p75NTR (extracellular) antibody (cat. no. ANT-007-AG, Alomone Labs, Jerusalem, Israel) (1:100).

Techniques: Expressing, Western Blot

Journal: Cell Reports Medicine

Article Title: An iPSC model of hereditary sensory neuropathy-1 reveals L-serine-responsive deficits in neuronal ganglioside composition and axoglial interactions

doi: 10.1016/j.xcrm.2021.100345

Figure Lengend Snippet: Neurotrophic signaling is impaired in HSN1 neurons (A) Photomicrographs of 8-week-old iPSCdSNs immunocytochemically stained for NF200 (green) and pERM (red) at baseline, after neurotrophic stimulation and after neurotrophic starvation. (B) Quantification of pERM membrane fluorescence intensity determined by profile plot analysis across multiple differentiations. Between groups comparison, ∗p ≤ 0.0001 by 2-way ANOVA (F = 31.51, df = 5). Individual comparisons by Tukey’s post hoc tests: healthy control baseline versus HSN1 baseline, ∗p = 0.0001; healthy control stimulated versus HSN1 stimulated, ∗p ≤ 0.0001; healthy control starved versus HSN1 starved, p = 0.939; and healthy control baseline versus healthy control starved, ∗p = < 0.0001. Each data point represents the mean from an independent differentiation and iPSC line (three iPSC lines analyzed across two differentiations per genotype). (C) Differential interference contrast (DIC) brightfield images of live control and HSN1 iPSCdSNs incubated with a fluorescently tagged anti-p75NTR antibody. (D) Quantification of p75NTR membrane fluorescence intensity determined by profile plot analysis across multiple differentiations. Control versus HSN1 comparison, ∗p = 0.0003 by 2-way ANOVA (F = 38.25, df = 1). Each data point represents the mean from an independent differentiation and iPSC line (three iPSC lines analyzed across two differentiations per genotype). (E) Western blots of 8-week-old iPSCdSNs for ERK and p-ERK with (+NT) and without (−NT) neurotrophic stimulation. (F) Normalized relative intensity of western blots expressed as p-ERK/ERK ratio for each control and HSN1 line, and mean averages (−NT, ∗p = 0.0166 and +NT, ∗p = 0.0008 by Student’s t test). Colors on graphs represent different iPSC lines and all error bars are SEM. All images within a panel are taken at the same magnification.

Article Snippet: Rabbit anti-P75NTR-ATTO-488 , Alomone Labs Cat# ANT-007-AG , RRID: AB_2341009.

Techniques: Staining, Fluorescence, Incubation, Western Blot

Journal: Cell Reports Medicine

Article Title: An iPSC model of hereditary sensory neuropathy-1 reveals L-serine-responsive deficits in neuronal ganglioside composition and axoglial interactions

doi: 10.1016/j.xcrm.2021.100345

Figure Lengend Snippet:

Article Snippet: Rabbit anti-P75NTR-ATTO-488 , Alomone Labs Cat# ANT-007-AG , RRID: AB_2341009.

Techniques: Labeling, Recombinant, Expressing, Software

Journal: bioRxiv

Article Title: Live cell imaging of single neurotrophin receptor molecules on human neuron in Alzheimer’s disease

doi: 10.1101/2020.02.17.953174

Figure Lengend Snippet: Intensity profiles of a single Atto-633-labeled TrkA (A) and Atto-488-labeled p75 NTR (B) signal. Histograms show the intensity value of every spot for TrkA (C) and p75 NTR (D) in a recording superimposed with a single-fitted Gaussian curve (blue line).

Article Snippet: Neurons were incubated with Atto-633 labeled anti-TrkA antibody (anti-TrkA (extracellular)-Atto-633, 1:100, Alomone Labs) or Atto-488 labeled anti-p75 NTR antibody (anti-p75 NTR (extracellular)-Atto-488,1:100, Alomone Labs) for 6 minutes at 37°C.

Techniques: Labeling