Journal: ACS Chemical Neuroscience

Article Title: Neurotropic activity and safety of methylene-cycloalkylacetate (MCA) derivative 3-(3-allyl-2-methylenecyclohexyl) propanoic acid

doi: 10.1021/acschemneuro.0c00255

Figure Lengend Snippet: HU- MCA-13 induced neurotropic effect in different neuronal cultures. Neurotropic effect was measured after treatment with 5 μM of HU-MCA-13 of rat PC12 dopaminergic neurons (7 days, top row), mice dorsal root ganglion (DRG) explants (5 days, middle row) and rat spinal cord sensory neurons (14 days, bottom row). The negative control cultures were treated with 0.1% DMSO and the positive control cultures were treated with 50 ng/mL mouse β-nerve growth factor (NGF) for the same periods of time.

Article Snippet: Tissue culture grade mouse β-NGF, was purchased from Alomone Laboratories (Jerusalem, Israel).

Techniques: Negative Control, Positive Control

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Schematic diagram of the CRISPR-Cas9 screen process. b Genes identified by NGS were analyzed with the MAGeCK program and plotted based on the log 2 value of fold change of NGS reads and statistical significance (shown as log 10 value of RRA p -value and plotted as the y -axis). The genes involved in the GPI (glycosylphosphatidylinositol) biosynthetic pathway are colored red. c Schematic diagram of human TFPIβ (TFPI), a GPI-anchored protein with two BPTI/Kunitz protease inhibitor domains (K1 and K2). N, N-termini; C, C-termini. HeLa ( d ) or A549 ( e ) KO cells lacking TFPI, TFPI2 (a homolog of TFPI), PIGS, or PIGV were generated via the CRISPR-Cas9 approach. UGP2-KO cells were also analyzed as a control. Cells were exposed to recombinant TcdB4.2 for 24 h. Their CR 50 values are normalized to WT and plotted in a bar-chart ( f ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). g – i HeLa ( g ) or 5637 ( h ) cells overexpressing triple-HA-tagged TFPI, TFPI2, or mouse TFPI (mTFPI) via lentiviral transduction were exposed to TcdB4.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations. Their CR 50 values are normalized to WT and plotted in a bar-chart ( i ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). j Binding of TcdB4.2 (500 nM) to Fc-tagged TFPI, TFPI2, and mTFPI (immobilized onto capture biosensors) was examined using biolayer interferometry (BLI) assays. Fc-tagged extracellular domains of FZD2 (CRD2), SEMA6A, and IgG were used as controls. Representative sensorgrams from one of three independent experiments are shown. k HeLa cells were exposed to either TcdB4.2 alone (4 pM) or TcdB4.2 pre-incubated with Fc-tagged TFPI, TFPI2, or mTFPI at the indicated molar ratios (1:250 ~ 1:20,000) on ice for 1 h. The percentage of cell-rounding at 6 h incubation was plotted. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). l HeLa-WT, TFPI-KO, and TFPI2-KO cells were exposed to recombinant TcdB2.1, TcdB7.2, TcdB12.1, TcsL, or culture supernatants of C. difficile strains expressing TcdB10.1 or TcdB11.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations are shown in Supplementary Fig. . Their CR 50 values were normalized to WT and plotted here. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments). m – p HeLa-WT, two CSPG4 KO single clones (CSPG4#1 and CSPG4#8), and two CSPG4/TFPI double KO cells (CSPG4#1-TFPI-KO and CSPG4#8-TFPI-KO) were exposed to TcdB4.2 ( m ), TcdB2.1 ( n ), or TcdB2.2 ( o ) for 24 h. The percentages of rounded cells were plotted over toxin concentrations. Their relative CR 50 values are plotted in a bar-chart ( p ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). q HeLa cells overexpressing HA-tagged TFPI, TFPI2, or mTFPI via lentiviral transduction were exposed to recombinant TcdB2.1, TcdB2.2, TcdB7.2, TcdB12.1, TcsL, or culture supernatants of C. difficile strains expressingTcdB10.1 or TcdB11.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations and are shown in Supplementary Fig. . Their CR 50 values are normalized to WT and plotted here. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: CRISPR, Protease Inhibitor, Generated, Recombinant, Transduction, Binding Assay, Incubation, Expressing, Clone Assay

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Schematic diagrams of TcdB4.2, TcdB4.2 1286–1805 , TcdB4.2 1835–2367 , TcdB1.1-FBD. TcdB4.1(B1.1) and TcdB1.1(B4.2) represent two mutant fragments exchanging 29 residues in the region 1432– 1600 that differ between TcdB4.1 and TcdB1.1. The numbers indicate the position of amino acid residues. GTD, glucosyltransferase domain; CPD, cysteine protease domain; DRBD, delivery/receptor-binding domain; CROPs, combined repetitive oligopeptides. Binding of 500 nM TcdB4.2 1286– 1805 ( b ) or TcdB4.2 1835– 2367 ( c ) to Fc-tagged TFPI and mTFPI was examined using BLI assays. Fc-tagged TFPI2, CRD2, SEMA6A, and IgG were used as controls. Representative sensorgrams from one of three independent experiments are shown. d HeLa cells transiently transfected with TFPI, TFPI2, mTFPI, SEMA6A, or FZD2 were exposed to FLAG-tagged TcdB4.2 1286– 1805 (5 µg/mL) on ice for 60 min, washed, fixed, permeabilized, and subjected to immunostaining analysis. Expression of exogenous proteins was confirmed by detecting fused HA or 1D4 tag. Nuclei were labeled with DAPI (blue). Scale bar, 5 µm. Representative images were from one of three independent experiments. Binding of 500 nM TcdB1.1-FBD, TcdB4.2 1286– 1805 , TcdB4.2(B1.1), and TcdB1.1(B4.2) to Fc-tagged CRD2 ( e ) and TFPI ( f ) was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. g Schematic diagram of TFPIβ, TFPI-K1, and TFPI-K2 fragments. Sig, signal peptide; N, N-terminal domain; K1, BPTI/Kunitz inhibitor domain 1; L1, loop 1; K2, BPTI/Kunitz inhibitor domain 2; L2, loop 2; β, GPI anchor sequence for TFPIβ. h Binding of 500 nM TcdB4.2 1286-1805 to Fc-tagged TFPI, TFPI-K1, and TFPI-K2 was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. i , j TcdB4.2 binding to TFPI-K2 prevents TFPI-K2 binding to its natural ligand coagulation factor Xa (FXa). FXa (0.5 ng/mL) cleaves its fluorescently labeled substrate and generates increasing fluorescent signal (measured as relative light unit, RLU, y -axis) over time ( x -axis). FXa’s enzymatic activity was inhibited by TFPI-K2 ( i , 7.5 ng/mL). The inhibitory effect of TFPI was blocked by adding TcdB4.2 1286– 1805 in a dose-dependent manner (1:1, 1:3, or 1:10 molar ratio). Representative curve from one of three independent experiments is shown. FXa activity was quantified by measuring the slope of RLU curves (1–10 min) and plotted as a bar-chart ( j ). The same experiments were also carried out for TFPI-Fc and mTFPI-Fc, with their curves shown in Supplementary Fig. and quantification in ( j ). Error bars indicate mean ± s.d.; N = 3; *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Mutagenesis, Binding Assay, Transfection, Immunostaining, Expressing, Labeling, Sequencing, Coagulation, Activity Assay

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Cultured undifferentiated human enteroids (in growth medium), differentiated human rectoids (in differentiation medium), and mouse intestinal organoids were exposed to either TcdB4.2 alone (10 pM) or TcdB4.2 pre-incubated with Fc-tagged TFPI, TFPI2, or mTFPI (100 nM) for 8 h. PBS was used as control (Ctrl). Stars indicate the dissociated organoids with released luminal contents; arrows indicate shrunken organoids; scale bar, 50 µm. Representative images are from one of three independent experiments. b Experiments were carried out as described in panel ( a ). After exposure to toxin for 3 days, cell viability was measured using the MTT assay and plotted as a bar-chart. Error bars indicate ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). c , d Accumulation of fluid in the thoracic cavity occurred within 15 h after intraperitoneal injection of TcdB4.2 into mice (50 ng per 25 g bodyweight). Injection of TcdB4.2 pre-incubated with Fc-tagged TFPI or mTFPI at 1:2000 molar ratios showed less fluid accumulation. Co-injection of TcdB4.2 with Fc-tagged TFPI2 at 1:2000 molar ratio did not affect fluid accumulation. Injection of saline was included as a control. The range of boxes indicates ± s.e.m.; whiskers indicate ± s.d.; percentiles indicate median; **, p < 0.01 (Student’s t -test, two-sided). e Experiments were carried out as described in panel ( c ) and the edema in lung tissues was evaluated by calculating dry-to-wet weight ratios. TcdB4.2 reduced dry-to-wet weight ratio of lung tissue more than in the saline group. Co-injection of TcdB4.2 with TFPI-Fc or mTFPI-Fc prevented this reduction, whereas co-injection with TFPI2-Fc showed no protection from TcdB4.2. The range of boxes indicates ± s.e.m.; whiskers indicate ± s.d.; percentiles indicate median; *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). f , g The sensitivity of HUVECs transfected with siRNAs targeting TFPI to TcdB1.1 or TcdB4.2 was analyzed using the 24 h cell-rounding assay. HUVECs transfected with non-targeting scrambled siRNAs served as a control. Dose-response curves are plotted in ( f ), and their relative CR 50 are plotted in a bar chart ( g ). Error bars indicate ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01; NS not significant (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Cell Culture, Incubation, MTT Assay, Injection, Transfection

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Amino acids across all 206 known TcdB sequences and 6 TcsL sequences were aligned and visualized using a haplotype coloring algorithm we recently developed , showing variation patterns across TcdB members. The first sequence (TcdB1.1) is assigned black color, and all other sequences colored black if they share the same residues. Unique residues in the second sequence (TcdB2.1) are colored green, followed by unique residues in the third sequence (TcdB4.2) colored red. The region 1460 to 1626 is enlarged for TcdB2/4/7 members. A unique B4/B7-haplotype can be visualized with residues in red color, with their position marked. Residue A1518 is unique in TcdB7 members and is colored gray. b The sequences of TcdB2.11, TcdB2.22, TcdB7.2, and TcdB7.5 were analyzed using a sliding window comparison with TcdB1.1, 2.1, 4.2, and 7.1, revealing their recombination patterns. Below each sliding window plot is a graphical summary depicting the recombination pattern. The location surrounding the TFPI/FZD-binding site (specificity-determining region) is marked. HeLa-WT, TFPI-KO, and TFPI2-KO cells were exposed to recombinant TcdB7.1 ( c ) or the culture supernatant of C. difficile strain expressing TcdB7.9 ( d ) for 24 h. The percentages of round-shaped cells were plotted over toxin or supernatant dilutions. Their CR values are normalized to WT and plotted in a bar-chart ( e ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). HeLa cells overexpressing HA-tagged TFPI, TFPI2, or mTFPI via lentiviral transduction were exposed to recombinant TcdB7.1 ( f ) or the culture supernatant of C. difficile strain expressing TcdB7.9 ( g ) for 24 h. The percentages of round-shaped cells were plotted over toxin or supernatant dilutions. Their CR values were normalized to WT and plotted in a bar-chart ( h ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Sequencing, Binding Assay, Recombinant, Expressing, Transduction

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: Binding of 500 nM TcdB1.1-FBD, TcdB2.1 1285– 1804 , TcdB2.11 1286– 1805 , TcdB4.2 1286– 1805 , TcdB7.2 1286– 1805 , TcdB7.5 1286– 1805 , TcdB10.1 1285– 1804 , TcdB11.2 1285– 1804 , TcdB12.1 1285– 1804 , and TcsL 1285– 1804 to Fc-tagged TFPI ( a ), mTFPI ( b ), or CRD2 ( c ) was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. d , e HeLa-WT, FZD1/2/7-KO, CSPG4-KO, and UGP2-KO cells were exposed to the culture supernatant of a C. difficile strain expressing TcdB2.22 for 24 h. The percentages of round-shaped cells were plotted over supernatant dilution ( d ). The relative CR 50 values in different cell lines were normalized to the WT and plotted as a bar-chart ( e ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). f , g HeLa-WT, TFPI-KO, and TFPI2-KO cells were exposed to the culture supernatant of a C. difficile strain expressing TcdB2.22 for 24 h. The percentages of round-shaped cells were plotted over supernatant dilutions ( f ). The relative CR50 values in different cell lines were normalized to the WT and plotted as bar-chart ( g ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments). h There are six residues that are different between TFPI-binding TcdB4.2, 2.11, 7.9 versus TcdB2.1 and 7.2 (1495, 1505, 1509, 1547, 1596, and 1599, marked as MT site 1–6). Mutagenesis studies were performed to replace the indicated residues on TcdB2.1 1285-1804 with the corresponding residues found in TcdB4.2. The binding of the indicated mutant proteins (500 nM) to immobilized TFPI-Fc was analyzed using BLI assays. TcdB2.11 1286– 1805 and TcdB2.1 1285– 1804 were analyzed as controls. Representative sensorgrams from one of three independent experiments are shown. i – k A TcdB1.1-FBD-5M mutant proteins were generated by replacing five residues in TcdB1.1 with the corresponding residues found in TcdB4.2 (positions 1495, 1505, 1547, 1596, and 1599). The binding of this mutant protein to immobilized TFPI-Fc ( i ), mTFPI-Fc ( j ), and CRD2 ( k ) was analyzed using BLI assays. TcdB1.1-FBD and TcdB4.2 1285– 1804 were analyzed as controls. Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Binding Assay, Expressing, Mutagenesis, Generated

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Manipulating NGF affects P2X3 receptor-mediated currents. A, Representative examples of currents induced by α,β-meATP (black bar) on TG neurons cultured in control condition (left), after treatment with anti-NGF antibody (24 h, middle) or incubated with NGF (50 ng/ml, 24 h). B, Dose–response curves for α,β-meATP in control condition (filled circles; n = 16–24), after anti-NGF antibody treatment (filled triangles; n = 8–24; *p = 0.03), and after NGF treatment (open circles; n = 12; *p = 0.033). Current amplitudes (ΔIN) were normalized with respect to the value obtained with 10 μm α,β-meATP; p values were calculated with absolute values. Note that, despite the effect of NGF or anti-NGF antibody application on α,β-meATP-induced current amplitude, there is no difference in agonist EC50 values. C, Histogram of real-time RT-PCR experiments of TG culture mRNAs shows that NGF increases P2X3 neosynthesis (n = 3 experiments; *p = 0.03). D, Example of Western immunoblots of P2X3 receptor from total extracts of TG neurons in control (lane 1), in anti-NGF antibody (24 h; lane 2), or NGF (50 ng/ml, 24 h; lane 3). Bottom lanes show control loading with β-tubulin III. Histograms show P2X3 optical density values expressed in AUs in the different culture conditions normalized with respect to the β-tubulin III signal (mean values in AUs; n = 5 experiments; p > 0.05).

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques: Cell Culture, Incubation, Quantitative RT-PCR, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Manipulating NGF affects desensitization of currents induced by α,β-meATP. A, Representative examples of currents induced by applications of α,β-meATP (black bar) in control condition (left), after anti-NGF antibody treatment (middle), and after NGF treatment (right). Traces of 10 μm α,β-meATP-evoked currents recorded after a second agonist application (30 s interval; see arrows) are superimposed. Note the differential recovery from desensitization in the three conditions. B, Histograms show no change in the time constant values of the α,β-meATP-evoked current decay, used as an index of desensitization onset (τfast; n = 151, n = 56, and n = 46 for control, anti-NGF, and NGF, respectively). C, Histograms show that anti-NGF treatment reduces recovery (*p < 0.0001) from desensitization of P2X3-mediated currents, whereas NGF increases (*p < 0.001) this value (n = 99, n = 49, and n = 38 for control, anti-NGF, and NGF, respectively). D, Representative traces of Ca2+ transients show that, with paired α,β-meATP application (black dots, 30 s spaced), the second response is more depressed after anti-NGF treatment than in control. Neurons are identified by their responsiveness to KCl (50 mm, 1 s), which remains unchanged after anti-NGF treatment. E, Histograms showing the depressant action of anti-NGF treatment on the amplitude of Ca2+ transients induced by 10 μm α,β-meATP or 50 mm KCl. Control level is indicated by a dashed line. *p ≤ 0.05; n = 4–5 experiments (30–40 cells in each experiment).

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Phosphorylation state of P2X3 receptors is controlled by NGF. A, Example of P2X3 receptor immunoprecipitation (IP) detected in Western blot (WB) with anti-phospho-threonine antibody. P2X3 receptors from untreated TG cultures show constitutive threonine phosphorylation (lane 1). Anti-NGF-treated samples show a reduced level of P2X3 threonine phosphorylation (lane 2), which is restored after acute application of NGF (50 ng/ml, <15 min) (lane 3). Total P2X3 inputs derived from the same lysates and immunostained with anti-P2X3 antibody are also shown (bottom lanes). B, Histograms show mean values (optical density AUs) of phosphorylated P2X3 subunits obtained from anti-NGF (*p= 0.008; n = 8 experiments) or anti-NGF plus NGF (*p= 0.009; n = 8) experiments. C, Acute enhancement by NGF (50 ng/ml) of α,β-meATP-induced currents (top records) is prevented by the PKC inhibitor chelerythrine (1.5 μm in the patch pipette; bottom records). D, Histograms of changes in P2X3 subunit threonine phosphorylation after acute application of NGF (*p = 0.036; n = 9)

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques: Immunoprecipitation, Western Blot, Derivative Assay, Transferring

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Anti-NGF treatment increases expression of heteromeric P2X2/3 receptors. A, Histograms showing residual current amplitude in control (n = 187) and after anti-NGF (n = 59) or NGF (n = 70) treatment. *p < 0.05. B, Cumulative probability plot of the ratio Iresidual/Ipeak for control condition (filled circles; n = 224), anti-NGF antibody treatment (filled triangles; n = 79), and NGF treatment (open circles; n = 85). Note that antibody-treated, but not NGF-treated, neurons show larger Iresidual, indicative of higher contribution by heteromeric P2X2/3 receptors. The activity of heteromeric P2X2/3 receptors is estimated as the ratio between the amplitude of the steady-state Iresidual at the end of α,β-meATP application and the one of the peak current (Ipeak). C, Example of biotinylation experiments showing that NGF deprivation treatment increases the surface expression of the P2X2 subunit (top), although it does not affect the amount of surface P2X3 (bottom). Histograms indicate changes in P2X2 (black) or P2X3 (white) surface receptors measured with optical density values expressed in AUs (n = 6 or 3 experiments, respectively; *p = 0.005) and normalized with respect to the protein amount in control condition (dashed line). D, Confocal microscopy photographs of TG neurons in the control and after anti-NGF treatment show different distribution of P2X2 immunostaining. In the absence of NGF, TG neurons express P2X2 receptors on the cell surface or close by (2.5 ± 1.5 μm beneath the surface; n = 15). Scale bar, 10 μm. Histograms show the relative percentage of neurons showing diffused or ring-like P2X2 immunoreactivity in control or after anti-NGF treatment (n = 469 or 211, respectively; *p < 0.001). E, Immunopurification of heteromeric P2X2/3 receptors was obtained with chemical cross-linking treatment (DTSSP) of membrane proteins. TG neuron extracts are immunoprecipitated (IP) with anti-P2X3 antibody and immunoblotted using anti-P2X2 antibody (lanes 1, 2). P2X2 subunit (64 kDa) is immunopurified from anti-NGF antibody-treated extracts (lane 2, arrowhead) and not from control (lane 1; n = 5 experiments) after DTSSP cross-linking. Anti-NGF-treated extracts, without DTSSP cross-linking, do not show any P2X2 signal (lane 3). P2X2 immunoprecipitation detects a single band (performed as control; lane 4, arrowhead). WB, Western blot. F, Anti-NGF treatment increases the fraction of TG neurons double immunopositive for P2X2 and P2X3 subunits (*p = 0.023), whereas chronic NGF application does not change this value with respect to control (n = 4).

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques: Expressing, Activity Assay, Confocal Microscopy, Immunostaining, Immu-Puri, Immunoprecipitation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: The potentiating action of NGF on P2X3 receptor-mediated responses does not depend on CGRP. A, Examples of currents induced by α,β-meATP in TG neurons grown in control condition, in the presence of NGF (50 ng/ml, 24 h), in the presence of CGRP receptor antagonist BIBN4096BS (BIBN; 50 ng/ml, 24 h), or with NGF plus BIBN4096BS (24 h). B, Somatic size distribution of TG neurons immunostained with TrkA receptor antibody (filled columns) and labeled with CGRP-RITC (open columns) to investigate colocalization of NGF and CGRP receptors (filled, cross-hatched columns). Data are from ∼1000 cells (n = 3 independent experiments). IR, Immunoreactivity. C, Example of patch-clamp traces showing that CGRP application (1 μm, 1 h) to anti-NGF antibody-treated neurons increases α,β-meATP responses and accelerates recovery from desensitization (30 s interval between twin pulses). D, CGRP (1 μm, 1 h) potentiates α,β-meATP-evoked currents in control condition (n = 19; *p < 0.0001) and after anti-NGF treatment (n = 18; *p < 0.0001).

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques: Labeling, Patch Clamp

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Trigeminal nociception evoked by α,β-meATP is prevented by anti-NGF treatment. A, Histograms showing the time spent by mice in face-rubbing activity after subcutaneous injection of α,β-meATP (10 μl bolus of 10 mm solution) into the upper lip after pretreatment with anti-NGF or saline (sal). Each bar is the mean of six to eight animals observed for the first 15 min epoch. No significant changes in rubbing activity are observed during this period. All mice were pretreated 24 h earlier with saline (10 μl/g, i.p.) or anti-NGF antibody (300 ng/g, i.p.). B, In the 15–30 min epoch, a significant increase (*p = 0.005) in rubbing activity is observed after injection of α,β-meATP (n = 8). This algogenic effect of α,β-meATP is blocked (*p= 0.025 vs sham pretreatment) after anti-NGF pretreatment (n = 7).

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques: Activity Assay, Injection

Journal: Brain, behavior, and immunity

Article Title: Intermittent cytomegalovirus infection alters neurobiological metabolism and induces cognitive deficits in mice

doi: 10.1016/j.bbi.2023.12.033

Figure Lengend Snippet: Validation of the intermittent MCMV infection/aging model. (A) Schematic outlining the experimental design, re-infection timeline, and cohort collection timepoints. Weight of Mock- (grey) and MCMV-infected (red) mice at (B) 1- (N = 10/group) or (C) 12-MPI (N = 27 mock, N = 28 MCMV). Unpaired t -test. Mean ± SD. (D) Weight change at monthly intervals over the 12-month experiment. Weight was normalized to initial weight of mice at 2-months of age, 1 day prior to first treatment. Unpaired t -test. Mean ± SD. ELISA-derived concentration of MCMV-specific (E) IgM and (F) IgG from isolated plasma. Two-way repeated measures ANOVA followed by Sidak’s multiple comparisons test. All data expressed as mean ± SD. ** p < 0.01; *** p < 0.001, **** p < 0.0001. MCMV = murine cytomegalovirus, DPI = days post infection, MPI = months post infection, EU = ELISA units. Cartoon created using BioRender.

Article Snippet: Murine cytomegalovirus (MCMV) Smith strain (ATCC, VR-1399) was passaged in mice and quantified via plaque assay as previously described ( ; ).

Techniques: Biomarker Discovery, Infection, Enzyme-linked Immunosorbent Assay, Derivative Assay, Concentration Assay, Isolation, Clinical Proteomics

Journal: Brain, behavior, and immunity

Article Title: Intermittent cytomegalovirus infection alters neurobiological metabolism and induces cognitive deficits in mice

doi: 10.1016/j.bbi.2023.12.033

Figure Lengend Snippet: A neuroinflammatory state is induced with repeated MCMV infections. (A) Representative images of immunofluorescence staining in the hippocampus and striatum. Subregions include dentate gyrus (DG), cornu Ammonis subfields (CA1, CA2, CA3), and stratam radiatum (SR). (B) Immunofluorescence staining of GFAP (astrocytic activity) and Iba-1 (microglial activation) in the hippocampus of young adult mice following a single MCMV exposure (left pair of images) or middle aged mice following multiple MCMV infections (12 months following initial MCMV exposure; right pair of images) compared to age-matched mock infected animals. (C) Quantification of fluorescence intensity of GFAP staining and cell counts of Iba-1 + cells in the hippocampus. (D) Immunofluorescence staining of GFAP and Iba-1 in the striatum following MCMV infection in these same groups of mice. (E) Quantification of fluorescence intensity of GFAP staining and cell counts of Iba-1 + cells in the striatum. All data expressed as mean +/− the SEM. Scale bars are 300 um for images in A and 40 um for images in B and D. Statistical comparisons were made using unpaired t -test between mock- and MCMV-exposed groups at a given timepoint (1 month or 12 months post-infection). * p < 0.05, ** p < 0.01. *** p < 0.001. a. u. = arbitrary units, MPI = months post infection.

Article Snippet: Murine cytomegalovirus (MCMV) Smith strain (ATCC, VR-1399) was passaged in mice and quantified via plaque assay as previously described ( ; ).

Techniques: Immunofluorescence, Staining, Activity Assay, Activation Assay, Infection, Fluorescence

Journal: Brain, behavior, and immunity

Article Title: Intermittent cytomegalovirus infection alters neurobiological metabolism and induces cognitive deficits in mice

doi: 10.1016/j.bbi.2023.12.033

Figure Lengend Snippet: Blood brain-barrier permeability is altered with repeated MCMV infections. (A) Immunofluorescence staining of Claudin-5, Fibrinogen, and IgG in the hippocampus of young adult mice following a single MCMV exposure (left pair of images) or middle aged mice following multiple MCMV infections (12 months following initial MCMV exposure; right pair of images) compared to age-matched mock infected animals. (B) Quantification of fluorescence intensity of Claudin-5, Fibrinogen, and IgG staining in the hippocampus. (C) Immunofluorescence staining of Claudin-5, Fibrinogen, and IgG in the striatum following MCMV infection in these same groups of mice. (D) Quantification of fluorescence intensity of Claudin-5, Fibrinogen, and IgG staining in the striatum. All data expressed as mean +/− the SEM. Scale bars are 40 um for images in A and C. Statistical comparisons were made using unpaired t -test between mock- and MCMV-exposed groups at a given timepoint (1 month or 12 months post-infection). * p < 0.05, ** p < 0.01. a.u. = arbitrary units, MPI = months post infection.

Article Snippet: Murine cytomegalovirus (MCMV) Smith strain (ATCC, VR-1399) was passaged in mice and quantified via plaque assay as previously described ( ; ).

Techniques: Permeability, Immunofluorescence, Staining, Infection, Fluorescence

Journal: Brain, behavior, and immunity

Article Title: Intermittent cytomegalovirus infection alters neurobiological metabolism and induces cognitive deficits in mice

doi: 10.1016/j.bbi.2023.12.033

Figure Lengend Snippet: Intermittent MCMV infection alters the transcriptional profile of brain cells. Multiome libraries were generated and integrated. (A) UMAP clustering of brain cell types based on single cell expression. (B) Number of nuclei characterized per cell population. (C) The average changes in metabolic associated gene expression for each cell type relative to mock-infected samples. (D) Changes to chromatin accessibility of metabolic associated genes in endothelial population with respect to mock-infected samples.

Article Snippet: Murine cytomegalovirus (MCMV) Smith strain (ATCC, VR-1399) was passaged in mice and quantified via plaque assay as previously described ( ; ).

Techniques: Infection, Generated, Expressing, Gene Expression

Journal: Brain, behavior, and immunity

Article Title: Intermittent cytomegalovirus infection alters neurobiological metabolism and induces cognitive deficits in mice

doi: 10.1016/j.bbi.2023.12.033

Figure Lengend Snippet: VDAC1 expression in brain microvascular endothelial cells is increased with repeated MCMV infection. (A) Cartoon identifying markers representative of glycolysis and oxidative phosphorylation pathways. (B) Flow cytometry was used to determine brain cell population changes as a percentage of live cells isolated in mock- and MCMV-infected mice at 1 MPI and 12 MPI. N=5/condition. Expression of metabolic markers in BMECs at (C) 1 MPI and (D) 12 MPI. Graphs represent pooled data from n=5 mice per treatment. Samples compared using an unpaired t test. Mean ± the SD. *, p < 0.05. DPI = days post infection, MPI = months post infection, BMEC = brain microvascular endothelial cells, eNOS = endothelial nitric oxide synthase, GLUT1 = glucose transporter 1, VDAC1 = voltage dependent anion channel 1, TOMM20 = translocase of the outer mitochondrial membrane complex subunit 20, MFI = mean fluorescence intensity, a.u. = arbitrary units. Cartoon generated using BioRender.

Article Snippet: Murine cytomegalovirus (MCMV) Smith strain (ATCC, VR-1399) was passaged in mice and quantified via plaque assay as previously described ( ; ).

Techniques: Expressing, Infection, Phospho-proteomics, Flow Cytometry, Isolation, Membrane, Fluorescence, Generated

Journal: Brain, behavior, and immunity

Article Title: Intermittent cytomegalovirus infection alters neurobiological metabolism and induces cognitive deficits in mice

doi: 10.1016/j.bbi.2023.12.033

Figure Lengend Snippet: Mitochondrial changes and oxidative stress are detected in BMV intermittently exposed to MCMV. (A) Cartoon illustrating how mitochondrial function was measured in BMV using the Seahorse XFe24 Mito Stress Kit. All samples were normalized to protein concentration. (B) Basal Respiration, Proton Leak, Non-Mitochondrial Oxygen Consumption. N=4, Unpaired t- test. (C) Mitochondrial mass was determined by flow cytometry using nonyl acridine orange (NAO) dye. N=4 Unpaired t- test. (D) Superoxide production was assessed using MitoSOX Red. N=4 Unpaired t test. All data expressed as mean ± SD. *, p < 0.05; **, p < 0.01. OCR = oxygen consumption rate, MPI = months post infection.

Article Snippet: Murine cytomegalovirus (MCMV) Smith strain (ATCC, VR-1399) was passaged in mice and quantified via plaque assay as previously described ( ; ).

Techniques: Protein Concentration, Flow Cytometry, Infection

Journal: Brain, behavior, and immunity

Article Title: Intermittent cytomegalovirus infection alters neurobiological metabolism and induces cognitive deficits in mice

doi: 10.1016/j.bbi.2023.12.033

Figure Lengend Snippet: Cognition is altered with intermittent MCMV infection. (A) Schematic describing correct versus incorrect alternations in the Y-maze. Quantification of number of total alternations and percentage of spontaneous (correct) alternations between Mock and MCMV mice at (B) 1 MPI and (C) 12 MPI timepoints. (D) Diagram presenting our proposed model of pathogen-associated accelerated cognitive aging. Mean ± SEM. Statistical comparisons were made using an unpaired t- test. * p < 0.05; MCMV = murine cytomegalovirus, MPI = months post infection.

Article Snippet: Murine cytomegalovirus (MCMV) Smith strain (ATCC, VR-1399) was passaged in mice and quantified via plaque assay as previously described ( ; ).

Techniques: Infection