rabbit anti adam10  (OriGene)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet: ADAM10 is strongly enriched at presynaptic sites. a Scheme of ADAM10 at the synaptic membrane with indicated C-terminal anti-ADAM10 antibody binding. Nt N-terminus, Ct C-terminus. b – d Validation of the C-terminal ADAM10 antibody. b Immunocytochemistry for ADAM10 in wildtype (+ / + , wt) and knockout (-/-, KO) MEF cells. Representative widefield image ( b 1 ) and quantification ( b 2 ) show strong reduction of ADAM10 immunoreactivity in KO MEF cells. n = 10 (wt), n = 11(KO) images from 1 MEF cell preparation. 2-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. c Immunoblot analysis of ADAM10 wt and KO mouse embryonic fibroblast (MEF) cell lysates, detected with C-terminal ADAM10 antibody, indicates loss of ADAM10 bands corresponding to the precursor and mature form of the protease in KO cells. pA10: precursor of ADAM10; mA10: mature ADAM10. d Immunoblot of P21 A10 cKO and wt cortical extracts shows strong reduction in the ADAM10 signal in the cKO. pA10: precursor of ADAM10; mA10: mature ADAM10. e Representative maximum projections of confocal images of hippocampal primary cultures at div3. Immunostaining for ADAM10 (green), the axonal marker tau (blue), and MAP2 (red) as a dendritic marker. Note the strong enrichment of ADAM10 at the axon and axonal growth cones already in young cultures. f Left: Representative maximum projection of confocal images of a div18 primary rat hippocampal neuron, transfected with a maxGFP cell fill (green) and stained for ADAM10 (red) and the presynaptic vesicle marker synaptophysin (blue) in an axon and at a dendrite. ADAM10 is present at presynaptic boutons. Note that dendritic spines are largely devoid of ADAM10. Right: Line scans of indicated axonal bouton and dendritic spine. g Representative gated STED images of mature rat hippocampal primary neurons (div17) stained for ADAM10 (green), in combination with presynaptic cytomatrix of the active zone (CAZ) protein piccolo (red) and the vesicle marker synaptophysin (blue) or the presynaptic CAZ protein bassoon (blue), and the postsynaptic scaffold shank3 (red). Boxes indicate position of zoom-ins, lines were used for the line profiles shown. Note the localization of ADAM10 on the presynaptic (bassoon, blue) site. Right: Line scans of indicated synapses. See also Figure

Article Snippet: Anti-ADAM10 (rabbit) , Abcam , Cat#ab1997; RRID:AB_302747.

Techniques: Membrane, Binding Assay, Immunocytochemistry, Knock-Out, Western Blot, Immunostaining, Marker, Transfection, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet: ADAM10 in enriched in vesicles of mossy fiber boutons, that show only minor morphological changes in cKO animals. a DAB staining of ADAM10 in an adult wildtype mouse hippocampus shows strong enrichment of ADAM10 in mossy fibers. DG dentate gyrus, MF Mossy fibers. b High magnification of ADAM10 DAB and control (without primary antibody) staining in MF-CA3 synapses. Note the strong ADAM10 localization to the presynaptic site (pink arrow) and lack of DAB staining at the presynaptic membrane in the control (yellow arrowhead). S: dendritic spine. B: mossy fiber bouton (false coloured in blue). c Immunogold EM of a P21 wildtype mouse with focus on hippocampal mossy fiber boutons. Note that lack of gold particles at the synaptic membrane and that gold particles localize to the outside of vesicles, as the antibody detects ADAM10’s cytosolic C-terminus (see the scheme). d Example of ADAM10 cKO and wt mossy fiber bouton 3D reconstructions from SBEM data. See also Videos S1–S4. e Quantification of MFB volume, surface area and sphericity. n = 19 (wt), n = 23 (cKO) boutons from 3 animals each. 2-tailed unpaired Student’s t-test (volume, surface area) and Mann Whitney U-test (sphericity). Data are represented as mean ± SEM. See also Figure

Article Snippet: Anti-ADAM10 (rabbit) , Abcam , Cat#ab1997; RRID:AB_302747.

Techniques: Staining, Control, Membrane, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet: ADAM10 is required for the expression of presynaptic mossy fiber short-term plasticity which does not depend on the enzymatic activity of the protease. a Image of an acute hippocampal slice with indicated positions for stimulating (MF) and recording (CA3) electrodes. b – d Mossy fiber plasticity of ADAM10 wt and cKO animals. b Paired pulse facilitation ratio at different stimulation frequencies and example traces (average of 5 sweeps, inlet) of evoked fEPSPs at 20 Hz of ADAM10 cKO and wt mice. ADAM10 cKO show impaired facilitation. n = 18 (wt), n = 19 (cKO) slices from 5 mice each. Two-way repeated measures ANOVA. ****p < 0.0001. Data are represented as mean ± SEM. c Example traces of train facilitation at 20 Hz in wt and cKO slices. Application of the group II mGluR agonist DCGIV (1 µM) leads to a loss of response and is used to prove the mossy fiber origin of the detected signal. d Quantification of the ratio calculated from the fEPSP amplitudes measured in response to train facilitation. ADAM10 cKO slices show an impaired response to train stimulation at 20 Hz (dark colours) and 10 Hz (light colours). 2-way repeated measures ANOVA. ****p < 0.0001. n = 18 slices (wt), n = 19 slices (cKO) from 5 mice each. Data are represented as mean ± SEM. e – h MF plasticity of wt animals with or without ADAM10 inhibitor (GI254023X) treatment. e Paired pulse facilitation ratio at different stimulation frequencies and example traces [average of 3 (GI254023X) or 4 (ctr) sweeps, inlet] of evoked fEPSPs at 20 Hz of wt mouse slices upon inhibition of ADAM10 activity. Application of the ADAM10 inhibitor GI254023X does not affect synaptic facilitation. Two-way repeated measures ANOVA. Treatment p = 0.3113. n = 12 slices (control); n = 11 slices (GI254023X) from 3 mice each. Data are represented as mean ± SEM. f Example traces of train facilitation at 20 Hz in wt slices with and without ADAM10 inhibitor (GI254023X). Application of the group II mGluR agonist DCGIV (1 µM) leads to a loss of response and is used to prove the mossy fiber origin of the detected signal. g Quantification of the ratio calculated from the fEPSP amplitudes measured in response to train facilitation. Treatment with ADAM10 inhibitor does not change the ratio calculated in response to train stimulation at 20 Hz (dark colours) and 10 Hz (light colours). Two-way repeated measures ANOVA. p = 0.5764 (10 Hz), p = 0.9124 (20 Hz). n = 12 (control); n = 11 slices (GI254023X) from 3 mice each. Data are represented as mean ± SEM. h Immunoblot analysis of acute hippocampal wt slices untreated or treated with GI254023X confirming that the application of the ADAM10 inhibitor does in fact reduce ADAM10 activity. Note the reduced substrates cleavage (PrPc to shed PrPc, N-cadherin to C-terminal fragment CTF) in the GI254023X group. d di-glycosylated, m mono-glycosylated, u unglycosylated. See also Figure

Article Snippet: Anti-ADAM10 (rabbit) , Abcam , Cat#ab1997; RRID:AB_302747.

Techniques: Expressing, Activity Assay, Inhibition, Control, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet: ADAM10 acts via the syt7 pathway. a Syt7 levels in hippocampus of ADAM10 cKO mice are reduced, while the major mossy fiber calcium buffer calbindin and the vesicle marker VAMP are unchanged. n = 3 acute slice preparations of 3 animals (same slices as in Fig. 4). Unpaired, 2-tailed Student’s test. b Synaptic syt7 associates with ADAM10 in a Ca 2+ -independent manner. Endogenous Co-immunoprecipitations from mouse synaptosomes in presence of 200 µM CaCl 2 or 2 mM EGTA. *Unspecific band. Note the different exposure times for the lower blot. syso: synaptosomes; ctr: control. c Heterologous Co-immunoprecipitations from Neuro-2a cells shows syt7-GFP is in one complex with ADAM10 in both calcium (200 µM) and calcium-free (2mM EGTA) conditions. d , e ADAM10 and syt7 co-localize in primary hippocampal cultures. d Representative maximum projections of STED Xtend superresolution images of mature primary hippocampal cultures stained with a CF488A-pre-labelled ADAM10 antibody (green), syt7 (red) and the presynaptic scaffold bassoon (blue) and line scans. e Proximity ligation assay (PLA) for ADAM10 and syt7 in mature hippocampal cultures showing PLA signals at neuronal processes/axons indicating that both proteins are in close proximity (< 40 nm). Example maximum projection of a spinning disc confocal image showing the PLA signal (blue), cell morphology marker actin (phalloidin, green) and the synaptic marker bassoon (red). See also Figure

Article Snippet: Anti-ADAM10 (rabbit) , Abcam , Cat#ab1997; RRID:AB_302747.

Techniques: Marker, Control, Staining, Proximity Ligation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet: ADAM10 C-terminus is required for mossy fiber short-term plasticity. a Scheme of tat-peptide in relation to ADAM10 C-terminus. l length, aa amino acids. b Paired pulse facilitation ratio at different stimulation frequencies and example traces (average of 5 sweeps, inlet) of evoked fEPSPs at 20 Hz in hippocampal slices treated with ctr-tat or ADAM10-tat peptide. Application of ADAM10 C-terminus targeted tat-peptide leads to impairment in facilitation. Two-way repeated measures ANOVA. **p = 0.0086. n = 12 slices from 3 mice each. Data are represented as mean ± SEM. c Example traces of train facilitation at 20 Hz in both experimental groups. Application of the group II mGluR agonist DCGIV (1 µM) is used as control for the mossy fiber origin of the detected signals. D Quantification of the ratio calculated from the fEPSP amplitudes measured in response to train facilitation. Impaired response upon ADAM10-tat-peptide treatment to train stimulation at 20 Hz (dark colours) and 10 Hz (light colours). 2-Way repeated measures ANOVA. * p = 0.0294 (10 Hz), * p = 0.0409 (20 Hz). n = 12 slices from 3 mice each. Data are represented as mean ± SEM. e Syt7 levels are not changed in hippocampal slices after treatment with ADAM10-tat peptide compared to control-tat peptide. n = 3 acute slice preparations of 3 animals. Unpaired, 2 -tailed Student’s test. See also Figure

Article Snippet: Anti-ADAM10 (rabbit) , Abcam , Cat#ab1997; RRID:AB_302747.

Techniques: Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet:

Article Snippet: Anti-ADAM10 (rabbit) , Abcam , Cat#ab1997; RRID:AB_302747.

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, Control, In Situ, Knock-Out, Software, Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: The mature active form of ADAM10 is increased in the HD mouse hippocampus and causes N-CAD proteolysis. A Representative Western blot for the mature active form of ADAM10 (m-ADAM10) in synaptosomal fractions obtained from the hippocampus of R6/2 transgenic mice and zQ175 heterozygous knock-in mice. β-III Tubulin, loading control. B Quantification of data shown in A. WT and R6/2 mice at 10–12 weeks of age: n=12–13 mice/genotype. WT and zQ175 mice at 54 weeks of age: n=9 mice/genotype. Data are represented as mean ± SEM. ****P < 0.0001, unpaired t test. C Representative Western blot of N-CAD-CTF in the hippocampus from WT and HD mice (R6/2 and zQ175). α-Tubulin, loading control. D Quantification of results shown in C. The N-CAD-CTF signal intensity has been divided for the FL N-CAD content, which has been determined by dividing FL N-CAD intensity over the α-Tubulin intensity. WT and R6/2 mice at 10–12 weeks of age: n=8–9 mice/genotype; WT and zQ175 mice at 54 weeks: n=3 mice/genotype. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, unpaired t test

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Western Blot, Transgenic Assay, Knock-In, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: ADAM10 heterozygous deletion in the forebrain rescues dendritic spine loss in the CA1 region of the hippocampus in R6/2 mice. A Representative examples of secondary apical dendritic segments of CA1 pyramidal neurons from 13-week-old WT, R6/2, R6/2-A10cKO and A10cKO mice. Scale bars: 10 µm, 80 × Objective. S, stubby spines; M, mushroom spines; T, thin spines. B Total dendritic spine density. C Stubby spine density. D Mushroom spine density. E Thin spine density. In B-E n = 3 mice/genotype were analyzed for a total of n = 30 neurons/genotype. Each dot in the graphs represents the mean ± SEM of the spine density in 10 µm dendrite for each neuron analyzed. *P < 0.05, ****P < 0.0001, One-way ANOVA with Tukey’s post hoc test

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: ADAM10 heterozygous deletion in the forebrain rescues ultrastructural defects of the HD hippocampal synapse. A Diagram showing SVs classification based on distance from the presynaptic membrane (docked: 0–50 nm, reserve: 50–300 nm, resting: > 300 nm) with corresponding tenuous background colors added as a guide for the eye in TEM images reported in panel ( B ). B Representative TEM images of excitatory synapses in pyramidal neurons of the CA1 region of the hippocampus of WT, R6/2, R6/2-A10cKO and A10cKO mice at 13 weeks of age. Scale bars: 100 nm. PSD, post-synaptic density. C Density of total SVs. D Density of docked SVs. E Density of reserve SVs. F Density of resting SVs. In C-F, n = 3 mice/genotype and n = 60 excitatory synapses/genotype were analyzed. Each dot in the graphs represents the n° SVs/µm 2 for each excitatory synapse analyzed. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Kruskal–Wallis with Dunn’s multiple comparisons test

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Membrane

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: ADAM10 heterozygous deletion in the forebrain enhances BDNF synthesis and promotes ERK phosphorylation in the R6/2 hippocampus. A Scheme of the mouse BDNF gene and BDNF mRNA isoforms. B Total BDNF mRNA level and level of BDNF mRNA isoforms in the hippocampus of WT, R6/2 and R6/2-A10cKO mice at 13 weeks of age. WT: n = 4–7; R6/2: n = 5–7; R6/2-A10cKO: n = 7–8. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA with Bonferroni’s post hoc test. For BDNF mRNA isoform II the forward and reverse primers (see Methods) led to simultaneous amplification of the transcript variant IIA, IIB, and IIC. C ELISA for BDNF in the hippocampus of WT, R6/2 and R6/2-A10cKO mice at 13 weeks of age. WT: n = 4; R6/2: n = 6; R6/2-A10cKO: n = 9. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, One-way ANOVA with Bonferroni’s post hoc test. D Representative Western blot for total and phosphorylated ERK1/2 in the hippocampus of WT, R6/2 and R6/2-A10cKO mice at 13 weeks of age. β-III Tubulin, loading control. E , F Quantification of data in D. WT: n = 5; R6/2: n = 8; R6/2-A10cKO: n = 11. Data are represented as mean ± SEM. **P < 0.01, ***P < 0.001, One-way ANOVA with Bonferroni’s post hoc test

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Amplification, Variant Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: TrkB mediates the neuroprotective effect determined by ADAM10 inhibition on long-lasting spine loss in HD hippocampal neurons. A Hippocampal neurons from WT and R6/2 mice were transfected at DIV5 with pcDNA3.1-mGreenLantern plasmid. The ADAM10 inhibitor GI254023X (GI, 1 µM) was administered from DIV6 until DIV14. The TrkB antagonist ANA12 (10 µM) was administered at DIV12 and cells were fixed at DIV14 for spine analyses and excitatory synapses quantification. B Immunofluorescence images of dendritic spines in hippocampal cultures: WT, WT + ANA12, R6/2, R6/2 + ANA12, R6/2 + GI; R6/2 + GI + ANA12. Scale bars: 10 µm. M, mushroom spines; T, thin spines; S, stubby spines. C-F Density of total, stubby, mushroom, and thin spines. Data are from n = 3 independent primary culture preparations. Each dot in the graphs represents the number of spines in a 100-µm-long dendrite. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, One-way ANOVA with Tukey’s post hoc test. G Immunofluorescence images of excitatory synapses in hippocampal cultures: WT, R6/2, R6/2 + GI; R6/2 + GI + ANA12. I Immunofluorescence images of excitatory synapses in hippocampal cultures: WT, zQ175, zQ175 + GI; zQ175 + GI + ANA12. Excitatory synapses in G and I were visualized by Bassoon/Homer1 immunostaining. Map2, pan neuronal marker. Upper panel scale bars: 50 μm; bottom panel scale bars: 10 μm. H , J Synapses quantification. Data are from n = 3 independent primary culture preparations. Each dot in the graphs represents the number of excitatory synapses in a 100-µm-long dendrite. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, One-way ANOVA with Tukey’s post-hoc test

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Inhibition, Transfection, Plasmid Preparation, Immunofluorescence, Immunostaining, Marker

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: Blocking active ADAM10 with GI254023X promotes LTP induction through the TrkB signaling pathway. A Experimental scheme of treatment of WT and R6/2 primary hippocampal neurons. The ADAM10 inhibitor GI254023X (GI, 1 µM) was administered from DIV6 until DIV14. The TrkB antagonist ANA12 (10 µM) was administered at DIV12 until DIV14. Chemical LTP was induced at DIV14 with 0.2 mM glycine for 15 min. For dendritic spine analyses hippocampal neurons were transfected at DIV5 with pcDNA3.1-mGreenLantern plasmid. B Representative traces of spontaneous EPSCs (sEPSCs) recorded at a holding potential of -70 mV in baseline condition and following chemical LTP-induction (cLTP) in primary hippocampal cell cultures obtained from WT and R6/2 mice. + GI and + ANA12 indicate the presence of these substances in culture medium and during electrophysiological recordings. C Graph comparing the amplitudes of sEPSCs in baseline condition and after cLTP induction in the different experimental conditions. Each dot corresponds to the value obtained from a single cell. Data are expressed as mean ± SEM and were analyzed by Two-way ANOVA with Bonferroni’s post hoc test. *P < 0.05, **P < 0.01. D Representative images of dendritic segments (mGreenLantern signal) and GluA1 immunostaining in basal condition and after cLTP induction. M, mushroom spines. Image crops of representative M spines were numbered from 1 to 6. Scale bars: 10 µm. E Quantification of mushroom spine density. Data are from n = 3 independent primary culture preparations. Each dot in the graph represents the number of mushroom spines in a 100-µm-long dendrite. Data are shown as % over the basal condition, which was set to 100, and are expressed as mean ± SEM. *P < 0.05, ***P < 0.001, unpaired t test. F Quantification of GluA1 signal. Data are from n = 3 independent primary culture preparations. Each dot in the graph represents GluA1 signal in a 100-µm-long dendrite. Data are expressed as mean ± SEM and were analyzed by Two-way ANOVA with Tukey’s post hoc test. P* < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. G Percentage of mushroom spines enriched in GluA1 in basal condition and after cLTP induction. Data are from n = 3–5 independent primary culture preparations. Each dot in the graph represents the number of mushroom spines in a 100-µm-long dendrite. Data are expressed as mean ± SEM and statistical analysis was performed by using Two-way ANOVA with Tukey’s post hoc test. P* < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Supplementary Table 1 and 2: detailed statistical outputs related to panel F and G

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Blocking Assay, Transfection, Plasmid Preparation, Immunostaining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: The ADAM10 and the BDNF/TrkB pathways at the HD hippocampal synapse. Defects in synaptic plasticity imply increased amounts of active ADAM10 in the HD hippocampus and downregulation of the BDNF/TrkB pathway. ADAM10 inhibition prevents the loss of long-lasting spines and enhances GluA1-AMPARs recruitment and LTP induction in mushroom spines, while also restoring BDNF and ERK signaling

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Inhibition