targeting peptide c  (Biosynth Carbosynth)

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: Custom tat-peptides targeting amino acids 709–730 of ADAM10 C-terminus (NP_031425.2) and control peptide (prolines changed to alanines) with > 95% purity (HPLC, mass spectrometry and solubility tested) were from Genosphere Biotechnologies (France).

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: Custom tat-peptides targeting amino acids 709–730 of ADAM10 C-terminus (NP_031425.2) and control peptide (prolines changed to alanines) with > 95% purity (HPLC, mass spectrometry and solubility tested) were from Genosphere Biotechnologies (France).

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: Custom tat-peptides targeting amino acids 709–730 of ADAM10 C-terminus (NP_031425.2) and control peptide (prolines changed to alanines) with > 95% purity (HPLC, mass spectrometry and solubility tested) were from Genosphere Biotechnologies (France).

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: Custom tat-peptides targeting amino acids 709–730 of ADAM10 C-terminus (NP_031425.2) and control peptide (prolines changed to alanines) with > 95% purity (HPLC, mass spectrometry and solubility tested) were from Genosphere Biotechnologies (France).

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: Custom tat-peptides targeting amino acids 709–730 of ADAM10 C-terminus (NP_031425.2) and control peptide (prolines changed to alanines) with > 95% purity (HPLC, mass spectrometry and solubility tested) were from Genosphere Biotechnologies (France).

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: Custom tat-peptides targeting amino acids 709–730 of ADAM10 C-terminus (NP_031425.2) and control peptide (prolines changed to alanines) with > 95% purity (HPLC, mass spectrometry and solubility tested) were from Genosphere Biotechnologies (France).

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

Journal: Acta biomaterialia

Article Title: Interplay Between Degradability and Integrin Signaling on Mesenchymal Stem Cell Function within Poly(ethylene glycol) Based Microporous Annealed Particle Hydrogels

doi: 10.1016/j.actbio.2019.11.009

Figure Lengend Snippet: Design and characterization of MAP hydrogels with varying degradability. a) A schematic of MAP hydrogels assembled from off-stoichiometric PEG microgels via a secondary thiol-ene photopolymerization. b) Peptide sequences designed as crosslinkers and cell-adhesive ligands to achieve varying degradability and integrin binding. c) A representative Z-stack projection image of MAP hydrogels labeled with Alexa Fluor 488- succinimidyl ester illustrating the internal porous structure. Scale bar is 100 μm. d) Degradation curves of non-deg, slow-deg and fast-deg MAP hydrogels in a 0.2 mg/mL collagenase solution at 37 °C. e) Storage modulus of slow-deg and fast deg MAP hydrogels after degradation with 0%, 33%, and 67% mass loss.

Article Snippet: The α5β1 integrin targeting peptide c(RRETAWA) was purchased from AAPPtec and was synthesized via an on-resin cyclization reaction of Ac-CAhxK(Alloc)RRETAWAE(ODmab), as previously described by Gandavarapu et al. [ 30 ].

Techniques: Binding Assay, Labeling

Journal: Acta biomaterialia

Article Title: Interplay Between Degradability and Integrin Signaling on Mesenchymal Stem Cell Function within Poly(ethylene glycol) Based Microporous Annealed Particle Hydrogels

doi: 10.1016/j.actbio.2019.11.009

Figure Lengend Snippet: The effect of degradability on hMSC spreading and proliferation in c(RRETAWA)-functionalized MAP hydrogels. a) Maximum intensity Z- projection of cytoskeleton staining of hMSCs cultured in MAP hydrogels after 2 and 8 days. Green represents F- actin and blue represents nuclei. Scale bars are 100 μm. b) Quantification of cell number. c) Quantification of cell volume. * comparison factor: degradability; # comparison factor: time. ** indicates p < 0.01, *** and ### indicate p < 0.0001, Two-way ANOVA and Tukey’s multiple comparisons test.

Article Snippet: The α5β1 integrin targeting peptide c(RRETAWA) was purchased from AAPPtec and was synthesized via an on-resin cyclization reaction of Ac-CAhxK(Alloc)RRETAWAE(ODmab), as previously described by Gandavarapu et al. [ 30 ].

Techniques: Staining, Cell Culture

Journal: Acta biomaterialia

Article Title: Interplay Between Degradability and Integrin Signaling on Mesenchymal Stem Cell Function within Poly(ethylene glycol) Based Microporous Annealed Particle Hydrogels

doi: 10.1016/j.actbio.2019.11.009

Figure Lengend Snippet: ECM proteins, collagen type I and fibronectin synthesized by hMSCs in MAP hydrogels with varying degradability and integrin-binding peptides after 8 days of culture. a) Z-projection images from confocal microscopy. Scale bars are 100 μm. b) Quantification of total ECM protein per 50 μL MAP hydrogels. * comparison factor: degradability; # comparison factor: integrin-binding peptide. * indicates p < 0.05, *** and ### indicate p < 0.0001, Two-way ANOVA by Tukey’s multiple comparisons test.

Article Snippet: The α5β1 integrin targeting peptide c(RRETAWA) was purchased from AAPPtec and was synthesized via an on-resin cyclization reaction of Ac-CAhxK(Alloc)RRETAWAE(ODmab), as previously described by Gandavarapu et al. [ 30 ].

Techniques: Synthesized, Binding Assay, Confocal Microscopy

Journal: Acta biomaterialia

Article Title: Interplay Between Degradability and Integrin Signaling on Mesenchymal Stem Cell Function within Poly(ethylene glycol) Based Microporous Annealed Particle Hydrogels

doi: 10.1016/j.actbio.2019.11.009

Figure Lengend Snippet: hMSC expression of OPG in RGDS and c(RRETAWA)-functionalized MAP hydrogels with varying degradability after 2, 5, and 8 days of culture. Three-way ANOVA results: time (p < 0.0001), time×degradability (p < 0.0001), integrin-binding peptide×degradability (p < 0.05).

Article Snippet: The α5β1 integrin targeting peptide c(RRETAWA) was purchased from AAPPtec and was synthesized via an on-resin cyclization reaction of Ac-CAhxK(Alloc)RRETAWAE(ODmab), as previously described by Gandavarapu et al. [ 30 ].

Techniques: Expressing, Binding Assay

Journal: Acta biomaterialia

Article Title: Interplay Between Degradability and Integrin Signaling on Mesenchymal Stem Cell Function within Poly(ethylene glycol) Based Microporous Annealed Particle Hydrogels

doi: 10.1016/j.actbio.2019.11.009

Figure Lengend Snippet: hMSC secretion of a, b) VEGF and c, d) BMP2 in MAP hydrogels functionalized with RGDS and c(RRETAWA) and with varying degradability after 8 days of culture. Data presented are both before and after normalization. * comparison factor: degradability; # comparison factor: integrin-binding peptide. * and # indicate p < 0.05, ## indicates p < 0.01, *** indicates p < 0.0001, Two-way ANOVA by Tukey’s multiple comparisons test.

Article Snippet: The α5β1 integrin targeting peptide c(RRETAWA) was purchased from AAPPtec and was synthesized via an on-resin cyclization reaction of Ac-CAhxK(Alloc)RRETAWAE(ODmab), as previously described by Gandavarapu et al. [ 30 ].

Techniques: Binding Assay