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R&D Systems lrp6 antibody
Lrp6 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/lrp6+af1505/Human+LRP-6+Antibody/pm24900428__ml300278f_si_001-107-16-19
Average 93 stars, based on 12 article reviews

lrp6 antibody - by Bioz Stars, 2026-09

93/100 stars

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Western Blot:

Article Title: Activation of Canonical Wingless-type MMTV Integration Site Family (Wnt) Signaling in Mature Adipocytes Increases ?-Catenin Levels and Leads to Cell Dedifferentiation and Insulin Resistance
Article Snippet: Total protein, 25–40 μg, was loaded on SDS-PAGE (Cambrex, Biosciences Inc.) gels for each sample. .. Immunoblots were performed with the following antibodies: Axin (H-98), WIF-1 (sc-25520), α-SMA (sc-32251), pY (99), and IR (sc-711) (all from Santa Cruz Biotechnology, Santa Cruz, CA); LRP6 (AF1505) and DKK1 (MAB 1765) (R&D Systems, Inc., UK); β-catenin ( {"type":"entrez-nucleotide","attrs":{"text":"C19220","term_id":"1631491","term_text":"C19220"}} C19220 ) and GSK3β ( {"type":"entrez-nucleotide","attrs":{"text":"G22320","term_id":"1342646","term_text":"G22320"}} G22320 ) (BD Transduction Laboratories); PKB (9272), PKB ser473 (9271), GSK3β ser9 (9331), IRS-1 (2382), pERK1/2 (9106), and β-catenin ser675 (9567) (all from Cell Signaling, New England Biolabs Ltd., UK); ERK1/2 (V803A, Promega Biotech AB, Stockholm, Sweden); PPARγ2 (MAB3630, Chemicon International, Inc., Millipore); and GSK3β Y214 (05–413, Upstate Biotechnology, Millipore AB, Sweden). ..

Image Search Results

Fig. 1. Expression of LRP6-Val induces synaptic defects in cultured neurons. (A) Diagram of canonical Wnt signaling. Left: In the absence of Wnt, β-catenin is se- questered and degraded by the destruction complex preventing transcription of Wnt target genes. Right: Wnt, LRP6, and Fz receptors form a complex. Activation of the pathway results in disheveled (Dvl) recruitment to the plasma membrane and disassembly of the destruction complex. β-Catenin accumulates and translocates to the nucleus enabling Wnt target gene transcription. (B) Schematic of LRP6 showing the location of the Lrp6-val SNP (red asterisk and arrow) and the areas to which the Wnt antagonist Dkk1 and Wnt ligands bind. (C) Confocal images of vGlut1 (red) puncta on isolated axons of neurons expressing enhanced green fluorescent protein (EGFP)– actin alone or EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar, 5 μm. (D) WT LRP6 promoted the assembly of presynaptic sites but LRP6-Val did not. N = 4 independent cultures, 10 to 12 axons per culture. Kruskal-Wallis with Dunn’s post hoc test. (E) Top: Confocal images of Homer1 (red) and GFP (green) of neurons ex- pressing EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar, 21 μm. Bottom: Higher-magnification images show dendritic spines (GFP; green) and Homer1 (red) puncta along dendrites. Scale bar, 5 μm. (F) Left: Expression of LRP6-Val reduced spine density. Three independent cultures, 8 to 10 cells per culture. One-way analysis of variance (ANOVA) with Tukey’s post hoc test. Right: Expression of LRP6-Val failed to increase spine size. N = 3 independent cultures, 8 to 10 cells per culture. Kruskal- Wallis with Dunn’s post hoc. (G) LRP6-Val expression led to smaller and fewer Homer1 puncta. N = 3 independent cultures, 8 to 10 images per culture. One-way ANOVA with Tukey’s post hoc test. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 1. Expression of LRP6-Val induces synaptic defects in cultured neurons. (A) Diagram of canonical Wnt signaling. Left: In the absence of Wnt, β-catenin is se- questered and degraded by the destruction complex preventing transcription of Wnt target genes. Right: Wnt, LRP6, and Fz receptors form a complex. Activation of the pathway results in disheveled (Dvl) recruitment to the plasma membrane and disassembly of the destruction complex. β-Catenin accumulates and translocates to the nucleus enabling Wnt target gene transcription. (B) Schematic of LRP6 showing the location of the Lrp6-val SNP (red asterisk and arrow) and the areas to which the Wnt antagonist Dkk1 and Wnt ligands bind. (C) Confocal images of vGlut1 (red) puncta on isolated axons of neurons expressing enhanced green fluorescent protein (EGFP)– actin alone or EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar, 5 μm. (D) WT LRP6 promoted the assembly of presynaptic sites but LRP6-Val did not. N = 4 independent cultures, 10 to 12 axons per culture. Kruskal-Wallis with Dunn’s post hoc test. (E) Top: Confocal images of Homer1 (red) and GFP (green) of neurons ex- pressing EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar, 21 μm. Bottom: Higher-magnification images show dendritic spines (GFP; green) and Homer1 (red) puncta along dendrites. Scale bar, 5 μm. (F) Left: Expression of LRP6-Val reduced spine density. Three independent cultures, 8 to 10 cells per culture. One-way analysis of variance (ANOVA) with Tukey’s post hoc test. Right: Expression of LRP6-Val failed to increase spine size. N = 3 independent cultures, 8 to 10 cells per culture. Kruskal- Wallis with Dunn’s post hoc. (G) LRP6-Val expression led to smaller and fewer Homer1 puncta. N = 3 independent cultures, 8 to 10 images per culture. One-way ANOVA with Tukey’s post hoc test. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

Article Snippet: 9, eabo7421 (2023) 13 January 2023 11 of 15 SC I ENCE ADVANCES | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 (Roche, 11867423001, RRID:AB_390918), Homer1, (Synaptic Systems, 160002, RRID:AB_2120990), Homer1, (Synaptic Systems, 160003, RRID:AB_887730), Homer1, (Synaptic Systems, 160006, RRID:AB_263122), LRP6 (Abcam, ab134146, RRID:AB_2895164), LRP6 (R&D Systems, AF1505, RRID:AB_2266025), LRP6 (Cell Signaling Technology, 2560, RRID:AB_2139329), LRP6 (Cell Signaling Technology, 3395, RRID:AB_1950408), pLRP6 (Cell Signaling Technology, 2568, RRID:AB_2139327), MAP2 (Abcam, ab5392, RRID:AB_2138153), MAP2 (Abcam, ab92434, RRID:AB_2138147), NeuN, (Cell Signaling Technology, 12943, RRID:AB_2630395), PSD-95 (Millipore, MAB1598, RRID:AB_94278), α-tubulin (Sigma-Aldrich, T9026, RRID:AB_477593), vGlut1 (Millipore, AB5905, RRID:AB_2301751), and vinculin (Sigma-Aldrich, V4505, RRID:AB_477617).

Techniques: Expressing, Cell Culture, Activation Assay, Clinical Proteomics, Membrane, Isolation

Fig. 2. Lrp6-val mice exhibit synaptic defects at 7 to 9 months. (A) Sanger trace examples of WT, Lrp6-val heterozygous (Lrp6-val het), and Lrp6-val homozygous (Lrp6- val hom) KI mice. (B) Dendritic spines were analyzed in Lrp6-val hom KI mice crossed to a Thy1-GFP line. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar, 100 μm. (C) Confocal images of apical dendrites of CA1 pyramidal neurons of WT and Lrp6-val mice at 7 to 9 months. Scale bar, 25 μm. Insets show spines along a dendrite. Scale bar, 3 μm. (D) Lrp6-val mice display reduced spine head width. WT, N = 3; Lrp6-val N = 4. Unpaired t test. *P < 0.05. (E) Representative paired-pulse recordings of synaptic currents from WT and Lrp6-val brain slices at different ISIs. (F) Graph displays the mean PPR from all recorded cells. Lrp6-val increased the PPR at 50-ms ISIs. N = 11 to 21 cells recorded from four to five animals per genotype. Repeated measures one-way ANOVA with Tukey’s post hoc test. *P < 0.05. Data are represented as means ± SEM.

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 2. Lrp6-val mice exhibit synaptic defects at 7 to 9 months. (A) Sanger trace examples of WT, Lrp6-val heterozygous (Lrp6-val het), and Lrp6-val homozygous (Lrp6- val hom) KI mice. (B) Dendritic spines were analyzed in Lrp6-val hom KI mice crossed to a Thy1-GFP line. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar, 100 μm. (C) Confocal images of apical dendrites of CA1 pyramidal neurons of WT and Lrp6-val mice at 7 to 9 months. Scale bar, 25 μm. Insets show spines along a dendrite. Scale bar, 3 μm. (D) Lrp6-val mice display reduced spine head width. WT, N = 3; Lrp6-val N = 4. Unpaired t test. *P < 0.05. (E) Representative paired-pulse recordings of synaptic currents from WT and Lrp6-val brain slices at different ISIs. (F) Graph displays the mean PPR from all recorded cells. Lrp6-val increased the PPR at 50-ms ISIs. N = 11 to 21 cells recorded from four to five animals per genotype. Repeated measures one-way ANOVA with Tukey’s post hoc test. *P < 0.05. Data are represented as means ± SEM.

Techniques:

Fig. 3. Lrp6-val mice display postsynaptic defects and impaired basal synaptic transmission, synaptic vesicle release, and RRP size at 12 months. (A) Top: Dendritic spines were analyzed at 12 to 14 months in Lrp6-val KI mice crossed to a Thy1-GFP line. Scale bar, 25 μm. Bottom: Confocal images of regions of interest containing apical dendrites of CA1 pyramidal neurons in WT and Lrp6-val mice. Scale bar, 3 μm. (B) Lrp6-val mice have smaller and fewer spines. WT, N = 4; Lrp6- val, N = 4. Unpaired t test. (C) Representative traces of EPSCs elicited at increasing stimulation voltages with an average of three responses for each stimulus voltage. (D) I/O curves showing a significant reduction in EPSC amplitude in hippocampal slices from Lrp6-val mice. N = 12 to 13 cells from four animals per genotype. Repeated measure one-way ANOVA with Tukey’s post hoc test. (E) Representative traces of paired pulse evoked EPSCs at different ISIs using brain slices from WT and Lrp6-val mice. (F) Graph displays the mean PPR from all cells. Lrp6-val mice display increased PPR at 50- and 100-ms ISI. N = 13 to 14 cells from four animals per genotype. Repeated measure one-way ANOVA with Tukey’s post hoc test. (G) Representative traces of EPSCs elicited by a 20-Hz electrical stimulation for 3 s recorded from WT and Lrp6-val mice. (H) Graph showing reduced mean cumulative charge in Lrp6-val mice. N = 12 cells from four animals per genotype. Repeated measure one-way ANOVAwith Tukey’s post hoc test. (I) Graph displays the RRP size, obtained from all cells. Lrp6-val mice exhibited a reduced RRP. N = 12 cells from four animals per genotype. Unpaired Student’s t test. Data are represented as means ± SEM. *P < 0.05 and ***P < 0.001.

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 3. Lrp6-val mice display postsynaptic defects and impaired basal synaptic transmission, synaptic vesicle release, and RRP size at 12 months. (A) Top: Dendritic spines were analyzed at 12 to 14 months in Lrp6-val KI mice crossed to a Thy1-GFP line. Scale bar, 25 μm. Bottom: Confocal images of regions of interest containing apical dendrites of CA1 pyramidal neurons in WT and Lrp6-val mice. Scale bar, 3 μm. (B) Lrp6-val mice have smaller and fewer spines. WT, N = 4; Lrp6- val, N = 4. Unpaired t test. (C) Representative traces of EPSCs elicited at increasing stimulation voltages with an average of three responses for each stimulus voltage. (D) I/O curves showing a significant reduction in EPSC amplitude in hippocampal slices from Lrp6-val mice. N = 12 to 13 cells from four animals per genotype. Repeated measure one-way ANOVA with Tukey’s post hoc test. (E) Representative traces of paired pulse evoked EPSCs at different ISIs using brain slices from WT and Lrp6-val mice. (F) Graph displays the mean PPR from all cells. Lrp6-val mice display increased PPR at 50- and 100-ms ISI. N = 13 to 14 cells from four animals per genotype. Repeated measure one-way ANOVA with Tukey’s post hoc test. (G) Representative traces of EPSCs elicited by a 20-Hz electrical stimulation for 3 s recorded from WT and Lrp6-val mice. (H) Graph showing reduced mean cumulative charge in Lrp6-val mice. N = 12 cells from four animals per genotype. Repeated measure one-way ANOVAwith Tukey’s post hoc test. (I) Graph displays the RRP size, obtained from all cells. Lrp6-val mice exhibited a reduced RRP. N = 12 cells from four animals per genotype. Unpaired Student’s t test. Data are represented as means ± SEM. *P < 0.05 and ***P < 0.001.

Techniques: Transmission Assay

Fig. 4. Presynaptic defects of Lrp6-val mice at 12 to 14 months. (A) Confocal images of vGlut1-labeled excitatory presynaptic terminals in the CA1 SR area of WT and Lrp6-val mice at 12 to 14 months. Scale bar, 2 μm. (B) Lrp6-val mice had fewer and smaller vGlut1 puncta. WT, N = 10; Lrp6-val, N = 9. Unpaired t test. *P < 0.05 and **P < 0.01. (C) EM images of an excitatory synapse of 12- to 14-month-old WT and Lrp6-val mice. Scale bar, 100 nm. (D) Lrp6-val mice had fewer synaptic vesicles, but no changes in PSD length were observed. N = 5, 19 to 25 images per animal. Mann-Whitney test. **P < 0.01. Data are represented as means ± SEM.

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 4. Presynaptic defects of Lrp6-val mice at 12 to 14 months. (A) Confocal images of vGlut1-labeled excitatory presynaptic terminals in the CA1 SR area of WT and Lrp6-val mice at 12 to 14 months. Scale bar, 2 μm. (B) Lrp6-val mice had fewer and smaller vGlut1 puncta. WT, N = 10; Lrp6-val, N = 9. Unpaired t test. *P < 0.05 and **P < 0.01. (C) EM images of an excitatory synapse of 12- to 14-month-old WT and Lrp6-val mice. Scale bar, 100 nm. (D) Lrp6-val mice had fewer synaptic vesicles, but no changes in PSD length were observed. N = 5, 19 to 25 images per animal. Mann-Whitney test. **P < 0.01. Data are represented as means ± SEM.

Techniques: Labeling, MANN-WHITNEY

Fig. 5. Lrp6-val mice exhibit synapse loss with age. (A, C, and E) Confocal images of the CA1 SR of WT and Lrp6-val mice labeled with Bassoon (green) and Homer1 (red) at 7 to 9 months (A), 12 months (C), and 16 to 18 months (E). Scale bars, 2.5 μm. Insets display high-magnification images of synapses. Scale bars, 2 μm. (B and D) Quantification of synapse number, based on the colocalization of pre- and postsynaptic puncta, showed no differences between WT and Lrp6-val mice at 7 to 9 months (B) or 12 months (D). N = 3 per genotype. Unpaired t test. (F) Synapse number was significantly reduced in Lrp6-val mice 16 to 18 months. WT, N = 8; Lrp6-val, N = 9. Unpaired t test. *P < 0.05. Data are represented as means ± SEM.

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 5. Lrp6-val mice exhibit synapse loss with age. (A, C, and E) Confocal images of the CA1 SR of WT and Lrp6-val mice labeled with Bassoon (green) and Homer1 (red) at 7 to 9 months (A), 12 months (C), and 16 to 18 months (E). Scale bars, 2.5 μm. Insets display high-magnification images of synapses. Scale bars, 2 μm. (B and D) Quantification of synapse number, based on the colocalization of pre- and postsynaptic puncta, showed no differences between WT and Lrp6-val mice at 7 to 9 months (B) or 12 months (D). N = 3 per genotype. Unpaired t test. (F) Synapse number was significantly reduced in Lrp6-val mice 16 to 18 months. WT, N = 8; Lrp6-val, N = 9. Unpaired t test. *P < 0.05. Data are represented as means ± SEM.

Techniques: Labeling

Fig. 6. Neurons from Lrp6-val mice fail to respond to Wnt7a and the presence of LRP6-Val affects the formation of the Wnt receptor complex and downstream signaling. (A) Diagram depicting hippocampal neuron isolation from WT and Lrp6-val mice. (B) Images of WT and Lrp6-val neurons treated with recombinant Wnt7a. vGlut1 (green), Homer1 (red), and MAP2 (Blue). Scale bar, 5 μm. (C) Wnt7a (200 ng/ml) increased synapse number in WT neurons but not in Lrp6-val neurons. N = 4 independent cultures. Two-way ANOVA with Games-Howell post hoc test. (D) Schematic of proximity ligation assay (PLA) to detect LRP6 and Fz5-HA interaction in close proximity (<40 nm). (E) Confocal images of HeLa cells expressing GFP (control), Fz5-HA, and WT LRP6 or LRP6-Val treated with control vehicle (Veh) or Wnt7a. GFP, green; PLA, red; and DAPI, blue. Scale bar, 10 μm. (F) The PLA signal intensity per cell was increased in cells expressing Fz5-HA and WT LRP6 or LRP6-Val compared to cells expressing GFP. N = 3 independent experiments. One-way ANOVA with Tukey’s post hoc test. (G) Wnt7a increased the PLA signal in cells expressing WT LRP6 and Fz5-HA but not in cells expressing LRP6-Val and Fz5-HA. N = 3 independent experiments. Two-way ANOVA with Tukey’s post hoc test. (H) Wnt7a increased pLRP6 when normalized to total LRP6 in HeLa cells expressing WT LRP6 and Fz5-HA but not in cells expressing LRP6-Val and Fz5-HA. WT LRP6 + Fz5-HA Veh, N = 50 cells; WT LRP6 + Fz5-HAWnt7a, N = 53 cells; LRP6-Val + Fz5-HAVeh, N = 71 cells; and LRP6-Val + Fz5-HAWnt7a, N = 61 cells from three independent experiments. Kruskal-Wallis with Dunn’s post hoc test. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. A.U. arbitrary units.

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 6. Neurons from Lrp6-val mice fail to respond to Wnt7a and the presence of LRP6-Val affects the formation of the Wnt receptor complex and downstream signaling. (A) Diagram depicting hippocampal neuron isolation from WT and Lrp6-val mice. (B) Images of WT and Lrp6-val neurons treated with recombinant Wnt7a. vGlut1 (green), Homer1 (red), and MAP2 (Blue). Scale bar, 5 μm. (C) Wnt7a (200 ng/ml) increased synapse number in WT neurons but not in Lrp6-val neurons. N = 4 independent cultures. Two-way ANOVA with Games-Howell post hoc test. (D) Schematic of proximity ligation assay (PLA) to detect LRP6 and Fz5-HA interaction in close proximity (<40 nm). (E) Confocal images of HeLa cells expressing GFP (control), Fz5-HA, and WT LRP6 or LRP6-Val treated with control vehicle (Veh) or Wnt7a. GFP, green; PLA, red; and DAPI, blue. Scale bar, 10 μm. (F) The PLA signal intensity per cell was increased in cells expressing Fz5-HA and WT LRP6 or LRP6-Val compared to cells expressing GFP. N = 3 independent experiments. One-way ANOVA with Tukey’s post hoc test. (G) Wnt7a increased the PLA signal in cells expressing WT LRP6 and Fz5-HA but not in cells expressing LRP6-Val and Fz5-HA. N = 3 independent experiments. Two-way ANOVA with Tukey’s post hoc test. (H) Wnt7a increased pLRP6 when normalized to total LRP6 in HeLa cells expressing WT LRP6 and Fz5-HA but not in cells expressing LRP6-Val and Fz5-HA. WT LRP6 + Fz5-HA Veh, N = 50 cells; WT LRP6 + Fz5-HAWnt7a, N = 53 cells; LRP6-Val + Fz5-HAVeh, N = 71 cells; and LRP6-Val + Fz5-HAWnt7a, N = 61 cells from three independent experiments. Kruskal-Wallis with Dunn’s post hoc test. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. A.U. arbitrary units.

Techniques: Isolation, Recombinant, Proximity Ligation Assay, Expressing, Control

Fig. 7. Lrp6-val exacerbates synapse loss around plaques in NL-G-F mice at 7 months. (A) Diagram shows synapse loss around an Aβ plaque (blue). (B) Confocal images of Bassoon (green) and Homer1 (red) at increasing distances from the center of an Aβ plaque (blue) in NL-G-F and NL-G-F;Lrp6-val mice or an equivalent point in WT and Lrp6-val mice, in the CA1 SR at 7 months. Scale bar, 4 μm. (C) NL-G- F mice displayed fewer synapses compared to WT mice or to Lrp6-val mice at 0 to 10 μm from the center of a plaque. Synapse number was reduced in NL-G-F;Lrp6- valmiceat 0 to 40 μm from the center of a plaque compared to NL-G-F mice. A sig- nificant reduction in synapse number was detected in NL-G-F;Lrp6-val micewhen compared to WT mice or Lrp6-val mice at all distances from the center of a plaque. WT = 17 slices from six animals, Lrp6-val = 18 slices from six animals, NL-G-F = 18 slices from seven animals, and NL-G-F;Lrp6-val = 19 slices from seven animals. Re- peated measure two-way ANOVA with Tukey’s post hoc test. *P < 0.05 and **P < 0.01. Data are represented as means ± SEM.

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 7. Lrp6-val exacerbates synapse loss around plaques in NL-G-F mice at 7 months. (A) Diagram shows synapse loss around an Aβ plaque (blue). (B) Confocal images of Bassoon (green) and Homer1 (red) at increasing distances from the center of an Aβ plaque (blue) in NL-G-F and NL-G-F;Lrp6-val mice or an equivalent point in WT and Lrp6-val mice, in the CA1 SR at 7 months. Scale bar, 4 μm. (C) NL-G- F mice displayed fewer synapses compared to WT mice or to Lrp6-val mice at 0 to 10 μm from the center of a plaque. Synapse number was reduced in NL-G-F;Lrp6- valmiceat 0 to 40 μm from the center of a plaque compared to NL-G-F mice. A sig- nificant reduction in synapse number was detected in NL-G-F;Lrp6-val micewhen compared to WT mice or Lrp6-val mice at all distances from the center of a plaque. WT = 17 slices from six animals, Lrp6-val = 18 slices from six animals, NL-G-F = 18 slices from seven animals, and NL-G-F;Lrp6-val = 19 slices from seven animals. Re- peated measure two-way ANOVA with Tukey’s post hoc test. *P < 0.05 and **P < 0.01. Data are represented as means ± SEM.

Techniques:

A) Diagram of canonical Wnt signalling showing the role of LRP6 in this pathway. The left panel shows that in the absence of Wnt, the LRP6 co-receptor does not form a complex with Frizzled receptors. β-catenin is sequestered and degraded by the destruction complex preventing transcription of Wnt target genes. The right panel shows that Wnt, LRP6 and Fz receptors form a complex required for downstream signalling. Activation of the pathway results in Dishevelled (Dvl) recruitment to the plasma membrane and disassembly of the destruction complex. β-catenin accumulates and translocates to the nucleus enabling transcription of Wnt target genes. B) Schematic representation of the LRP6 protein showing the location of the Lrp6-val SNP (red asterisk and arrow) and the areas where the Wnt antagonist Dkk1 and Wnt ligands bind to. C) Confocal images of vGlut1 (red) puncta on isolated axons of neurons expressing EGFP-actin alone or EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar = 5 μm. D) Quantification of vGlut1 puncta density showed that WT LRP6 promoted the assembly of presynaptic sites but LRP6-Val did not. N = 4 independent cultures, 10-12 axons imaged per culture. Kruskal Wallace with Dunn’s post-hoc test. * p < 0.05. E) Top: Confocal images of Homer1 (red) and GFP (green) of neurons expressing EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar = 21 μm. Bottom: Higher magnification of areas of interest show dendritic spines (GFP; green) and Homer1 (red) puncta along dendrites. Scale bar = 5 μm. F) Left panel: Gain-of-function of LRP6-Val resulted in reduced spine density. 3 independent cultures, 8-10 cells imaged per culture. One-way-ANOVA with Tukey’s post-hoc test. *** p < 0.001. Right panel: Expression of LRP6-Val failed to increase spine size. 3 independent cultures, 8-10 cells imaged per culture. Kruskal-Wallis with Dunn’s post-hoc. ** p < 0.01; *** p < 0.001. G) LRP6-Val expression led to smaller Homer1 puncta. 2 independent cultures, 8-10 cells imaged per culture. One-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. Data are represented as mean ± SEM.

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Diagram of canonical Wnt signalling showing the role of LRP6 in this pathway. The left panel shows that in the absence of Wnt, the LRP6 co-receptor does not form a complex with Frizzled receptors. β-catenin is sequestered and degraded by the destruction complex preventing transcription of Wnt target genes. The right panel shows that Wnt, LRP6 and Fz receptors form a complex required for downstream signalling. Activation of the pathway results in Dishevelled (Dvl) recruitment to the plasma membrane and disassembly of the destruction complex. β-catenin accumulates and translocates to the nucleus enabling transcription of Wnt target genes. B) Schematic representation of the LRP6 protein showing the location of the Lrp6-val SNP (red asterisk and arrow) and the areas where the Wnt antagonist Dkk1 and Wnt ligands bind to. C) Confocal images of vGlut1 (red) puncta on isolated axons of neurons expressing EGFP-actin alone or EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar = 5 μm. D) Quantification of vGlut1 puncta density showed that WT LRP6 promoted the assembly of presynaptic sites but LRP6-Val did not. N = 4 independent cultures, 10-12 axons imaged per culture. Kruskal Wallace with Dunn’s post-hoc test. * p < 0.05. E) Top: Confocal images of Homer1 (red) and GFP (green) of neurons expressing EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar = 21 μm. Bottom: Higher magnification of areas of interest show dendritic spines (GFP; green) and Homer1 (red) puncta along dendrites. Scale bar = 5 μm. F) Left panel: Gain-of-function of LRP6-Val resulted in reduced spine density. 3 independent cultures, 8-10 cells imaged per culture. One-way-ANOVA with Tukey’s post-hoc test. *** p < 0.001. Right panel: Expression of LRP6-Val failed to increase spine size. 3 independent cultures, 8-10 cells imaged per culture. Kruskal-Wallis with Dunn’s post-hoc. ** p < 0.01; *** p < 0.001. G) LRP6-Val expression led to smaller Homer1 puncta. 2 independent cultures, 8-10 cells imaged per culture. One-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. Data are represented as mean ± SEM.

Article Snippet: APP (6E10) (Novus Biotech, NBP2-62566), Amyloid-β (BioLegend, 803001), β-Actin (Cell Signalling Technology, 4970), Bassoon (Novus Biologicals, NB120-13249), GFP (Millipore, 06-896), GFP (Invitrogen, A-6455), Homer1, (Synaptic systems, 160002), Homer1, (Synaptic systems, 160003), LRP6 (Abcam, ab134146), LRP6 (R&D, AF1505), LRP6 (Cell Signalling Technology, 2560), LRP6 (Cell Signalling Technology, 3395), HA (Sigma, H6908), HA (Roche, 11867423001), MAP2 (Abcam, ab5392), MAP2 (Abcam, ab92434), NeuN, (Cell Signalling Technology, 12943), PSD-95 (Millipore, MAB1598), α-Tubulin (Sigma, T9026), vGlut1 (Millipore, AB5905), Vinculin (Sigma, V4505).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Isolation, Expressing

A) Sanger trace examples of WT, Lrp6-val heterozygous ( Lrp6-val het) and Lrp6-val homozygous ( Lrp6-val hom) knock-in mice. B) Dendritic spines were analysed in Lrp6-val hom knock-in mice crossed to a Thy1-GFP line. SO = stratum oriens, SP = stratum pyramidale, SR = stratum radiatum. Scale bar = 100 μm. C) Confocal images of apical dendrites of CA1 pyramidal neurons of WT and Lrp6-val mice at 7-9 months. Scale bar = 25 μm. Inset shows spines along a dendrite. Scale bar = 3 μm. D) Lrp6-val mice display reduced spine head width. WT N = 3 Lrp6-val N = 4. Unpaired T-test. * p < 0.05. E) Representative paired-pulse recordings of synaptic currents from WT and Lrp6-val brain slices at different inter-stimulus intervals (ISIs). F) Graph displays the mean PPR from all recorded cells. Lrp6-val increased the PPR a 50 ms inter-stimulus intervals. N = 11-21 cells recorded from 4-5 animals per genotype. Repeated measures one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. Data are represented as mean ± SEM.

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Sanger trace examples of WT, Lrp6-val heterozygous ( Lrp6-val het) and Lrp6-val homozygous ( Lrp6-val hom) knock-in mice. B) Dendritic spines were analysed in Lrp6-val hom knock-in mice crossed to a Thy1-GFP line. SO = stratum oriens, SP = stratum pyramidale, SR = stratum radiatum. Scale bar = 100 μm. C) Confocal images of apical dendrites of CA1 pyramidal neurons of WT and Lrp6-val mice at 7-9 months. Scale bar = 25 μm. Inset shows spines along a dendrite. Scale bar = 3 μm. D) Lrp6-val mice display reduced spine head width. WT N = 3 Lrp6-val N = 4. Unpaired T-test. * p < 0.05. E) Representative paired-pulse recordings of synaptic currents from WT and Lrp6-val brain slices at different inter-stimulus intervals (ISIs). F) Graph displays the mean PPR from all recorded cells. Lrp6-val increased the PPR a 50 ms inter-stimulus intervals. N = 11-21 cells recorded from 4-5 animals per genotype. Repeated measures one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. Data are represented as mean ± SEM.

Techniques: Knock-In

A) Top: Dendritic spines were analysed at 12-14 months in Lrp6-val knock-in mice crossed to a Thy1-GFP line. Scale bar = 25 μm. Bottom: Confocal images of regions of interest containing apical dendrites of CA1 pyramidal neurons in WT and Lrp6-val mice. Scale bar = 3 μm. B) Lrp6-val mice have smaller and fewer spines. WT N = 4, Lrp6-val N = 4. Unpaired T-test. * p < 0.05, *** p < 0.001. C) Representative traces of evoked excitatory post-synaptic currents (EPSCs) elicited at increasing stimulation voltages with an average of three responses for each stimulus voltage (10 V; 20 V; 30 V; 40 V and 50 V). D) Input-output curves showing a significant reduction in EPSC amplitude in hippocampal slices from Lrp6-val mice. N = 12-13 cells recorded from 4 animals per genotype. Repeated measures one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. E) Representative traces of paired pulse evoked EPSCs at different interstimulus intervals using brain slices from WT and Lrp6-val mice. F) Graph displays the mean PPR from all cells. Lrp6-val mice display increased PPR at 50 ms and 100 ms ISI. N = 13-14 cells from 4 animals per genotype. Repeated measure one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. G) Representative traces of EPSCs elicited by a 20 Hz electrical stimulation for 3 s recorded from WT and Lrp6-val mice. H) Graph showing reduced mean cumulative charge in Lrp6-val mice. N = 12 cells from 4 animals per genotype. Repeated measure one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. I) Graph displays the readily releasable pool (RRP) size, obtained from all cells. Lrp6-val mice exhibited a reduced RRP. N = 12 cells from 4 animals per genotype. Unpaired Student’s T-test. * p < 0.05. Data are represented as mean ± SEM.

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Top: Dendritic spines were analysed at 12-14 months in Lrp6-val knock-in mice crossed to a Thy1-GFP line. Scale bar = 25 μm. Bottom: Confocal images of regions of interest containing apical dendrites of CA1 pyramidal neurons in WT and Lrp6-val mice. Scale bar = 3 μm. B) Lrp6-val mice have smaller and fewer spines. WT N = 4, Lrp6-val N = 4. Unpaired T-test. * p < 0.05, *** p < 0.001. C) Representative traces of evoked excitatory post-synaptic currents (EPSCs) elicited at increasing stimulation voltages with an average of three responses for each stimulus voltage (10 V; 20 V; 30 V; 40 V and 50 V). D) Input-output curves showing a significant reduction in EPSC amplitude in hippocampal slices from Lrp6-val mice. N = 12-13 cells recorded from 4 animals per genotype. Repeated measures one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. E) Representative traces of paired pulse evoked EPSCs at different interstimulus intervals using brain slices from WT and Lrp6-val mice. F) Graph displays the mean PPR from all cells. Lrp6-val mice display increased PPR at 50 ms and 100 ms ISI. N = 13-14 cells from 4 animals per genotype. Repeated measure one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. G) Representative traces of EPSCs elicited by a 20 Hz electrical stimulation for 3 s recorded from WT and Lrp6-val mice. H) Graph showing reduced mean cumulative charge in Lrp6-val mice. N = 12 cells from 4 animals per genotype. Repeated measure one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. I) Graph displays the readily releasable pool (RRP) size, obtained from all cells. Lrp6-val mice exhibited a reduced RRP. N = 12 cells from 4 animals per genotype. Unpaired Student’s T-test. * p < 0.05. Data are represented as mean ± SEM.

Techniques: Knock-In

A) Confocal images of vGlut1-labelled excitatory presynaptic terminals in the CA1 stratum radiatum area of WT and Lrp6-val mice at 12-14 months. Scale bar = 2 μm. B) Lrp6-val mice had fewer and smaller vGlut1 puncta. WT N = 10 Lrp6-val N = 9. Unpaired T-test. * p < 0.05, ** p < 0.01. C) Electron microscopy images of an excitatory synapse of 12-14-month-old WT and Lrp6-val mice. Scale bar = 100 nm. D) Lrp6-val mice had fewer synaptic vesicles but no changes in PSD length were observed. N = 5, 19-25 images per animal. Mann-Whitney test. ** p < 0.01. Data are represented as mean ± SEM.

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Confocal images of vGlut1-labelled excitatory presynaptic terminals in the CA1 stratum radiatum area of WT and Lrp6-val mice at 12-14 months. Scale bar = 2 μm. B) Lrp6-val mice had fewer and smaller vGlut1 puncta. WT N = 10 Lrp6-val N = 9. Unpaired T-test. * p < 0.05, ** p < 0.01. C) Electron microscopy images of an excitatory synapse of 12-14-month-old WT and Lrp6-val mice. Scale bar = 100 nm. D) Lrp6-val mice had fewer synaptic vesicles but no changes in PSD length were observed. N = 5, 19-25 images per animal. Mann-Whitney test. ** p < 0.01. Data are represented as mean ± SEM.

Techniques: Electron Microscopy, MANN-WHITNEY

A, C, E) Confocal images of the CA1 stratum radiatum of WT and Lrp6-val mice labelled with Bassoon (green) and Homer1 (red) at 7-9 months (A), 12 months (C) and 16-18 months (E). Scale bar = 2.5 μm. Insets display high magnification images of synapses. Scale bar = 2 μm. B and D) Quantification of synapse number, based on the co-localisation of pre- and post-synaptic puncta, showed no differences between WT and Lrp6-val mice at 7-9 months (B) or 12 months (D). N = 3 per genotype. Unpaired T-test. F) Synapse number was significantly reduced in Lrp6-val mice 16-18 months. WT N = 8, Lrp6-val N = 9. Unpaired T-test. * p < 0.05. Data are represented as mean ± SEM.

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A, C, E) Confocal images of the CA1 stratum radiatum of WT and Lrp6-val mice labelled with Bassoon (green) and Homer1 (red) at 7-9 months (A), 12 months (C) and 16-18 months (E). Scale bar = 2.5 μm. Insets display high magnification images of synapses. Scale bar = 2 μm. B and D) Quantification of synapse number, based on the co-localisation of pre- and post-synaptic puncta, showed no differences between WT and Lrp6-val mice at 7-9 months (B) or 12 months (D). N = 3 per genotype. Unpaired T-test. F) Synapse number was significantly reduced in Lrp6-val mice 16-18 months. WT N = 8, Lrp6-val N = 9. Unpaired T-test. * p < 0.05. Data are represented as mean ± SEM.

Techniques:

A) Diagram depicting hippocampal neuron isolation from WT and Lrp6-val mice. B) Confocal images of WT and Lrp6-val hippocampal neurons treated with Wnt7a showing vGlut1 (green), Homer1 (red) and MAP2 (Blue). Scale bar = 5 μm. C) Quantification shows that Wnt7a increased the number of synapses in WT neurons, but Wnt7a had no effect on Lrp6-val neurons. N = 4 independent cultures. Two-way-ANOVA with Games-Howell post hoc test. * p < 0.05. D) Schematic of the PLA reaction: antibodies raised from two different species binds to the proteins of interest: LRP6 and Fz5-HA. Secondary antibodies, called PLA probes (+ and -), are linked to specific DNA strands. When proteins are in close proximity (<40 nm), DNA probes can hybridise. PCR amplification with fluorescent complementary probes allows the visualisation of the interaction by fluorescence microscopy. E) Confocal images of HeLa cells expressing GFP (control), WT LRP6 and Fz5-HA or LRP6 Val and Fz5-HA treated with control vehicle (BSA) or Wnt7a. GFP (green), PLA (red) and DAPI (Blue). Scale bar = 10 μm. F) Under basal conditions, the PLA signal intensity per cell was increased in cells expressing WT LRP6 and Fz5-HA or LRP6 Val and Fz5-HA compared to cells only expressing GFP. N = 3 independent experiments. One-way-ANOVA with Tukey’s post-hoc test. ** p < 0.01 and *** p < 0.001. G) Quantification shows that Wnt7a increased the PLA signal in cells expressing WT LRP6 and Fz5-HA but not in cells expressing LRP6 Val and Fz5-HA. N = 3 independent experiments. Two-way-ANOVA with Tukey’s post hoc test. * p < 0.05. Data are represented as mean ± SEM.

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Diagram depicting hippocampal neuron isolation from WT and Lrp6-val mice. B) Confocal images of WT and Lrp6-val hippocampal neurons treated with Wnt7a showing vGlut1 (green), Homer1 (red) and MAP2 (Blue). Scale bar = 5 μm. C) Quantification shows that Wnt7a increased the number of synapses in WT neurons, but Wnt7a had no effect on Lrp6-val neurons. N = 4 independent cultures. Two-way-ANOVA with Games-Howell post hoc test. * p < 0.05. D) Schematic of the PLA reaction: antibodies raised from two different species binds to the proteins of interest: LRP6 and Fz5-HA. Secondary antibodies, called PLA probes (+ and -), are linked to specific DNA strands. When proteins are in close proximity (<40 nm), DNA probes can hybridise. PCR amplification with fluorescent complementary probes allows the visualisation of the interaction by fluorescence microscopy. E) Confocal images of HeLa cells expressing GFP (control), WT LRP6 and Fz5-HA or LRP6 Val and Fz5-HA treated with control vehicle (BSA) or Wnt7a. GFP (green), PLA (red) and DAPI (Blue). Scale bar = 10 μm. F) Under basal conditions, the PLA signal intensity per cell was increased in cells expressing WT LRP6 and Fz5-HA or LRP6 Val and Fz5-HA compared to cells only expressing GFP. N = 3 independent experiments. One-way-ANOVA with Tukey’s post-hoc test. ** p < 0.01 and *** p < 0.001. G) Quantification shows that Wnt7a increased the PLA signal in cells expressing WT LRP6 and Fz5-HA but not in cells expressing LRP6 Val and Fz5-HA. N = 3 independent experiments. Two-way-ANOVA with Tukey’s post hoc test. * p < 0.05. Data are represented as mean ± SEM.

Techniques: Isolation, Amplification, Fluorescence, Microscopy, Expressing, Control

A) Diagram shows synapse loss around an Aβ plaque (blue). B) Confocal images of Bassoon (green) and Homer1 (red) at increasing distances from the centre of an Aβ plaque (blue) in NL-G-F and NL-G-F;Lrp6-val mice, or an equivalent point in WT and Lrp6-val mice, in the CA1 stratum radiatum at 7 months. Scale bar = 4 μm. C) NL-G-F mice displayed fewer synapses compared to WT mice or to Lrp6-val mice at 0-10 μm from the centre of a plaque. Synapse number was reduced in NL-G-F;Lrp6-val at 0-40 μm from the center of a plaque compared to NL-G-F mice. A significant reduction in synapse number was detected in NL-G-F;Lrp6-val when compared to WT mice or Lrp6-val mice at all distances from the center of a plaque. N = 6-7 brains per genotype and 2-3 slices per brain. Two-way-ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01. Data are represented as mean ± SEM.

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Diagram shows synapse loss around an Aβ plaque (blue). B) Confocal images of Bassoon (green) and Homer1 (red) at increasing distances from the centre of an Aβ plaque (blue) in NL-G-F and NL-G-F;Lrp6-val mice, or an equivalent point in WT and Lrp6-val mice, in the CA1 stratum radiatum at 7 months. Scale bar = 4 μm. C) NL-G-F mice displayed fewer synapses compared to WT mice or to Lrp6-val mice at 0-10 μm from the centre of a plaque. Synapse number was reduced in NL-G-F;Lrp6-val at 0-40 μm from the center of a plaque compared to NL-G-F mice. A significant reduction in synapse number was detected in NL-G-F;Lrp6-val when compared to WT mice or Lrp6-val mice at all distances from the center of a plaque. N = 6-7 brains per genotype and 2-3 slices per brain. Two-way-ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01. Data are represented as mean ± SEM.

Techniques:

Fig. 2. VAP1 cleaves LRP6 at the activation region. (A) HeLa cells were incubated with or without 0.4 lgmL1 VAP1 in serum-free medium for 2 h at 37 °C. The cells (1.5 9 107 cells) were harvested with a lysis buffer (25 mM Tris/HCl, pH 7.4, 300 mM NaCl, 1.5 mM MgCl2, 0.5% Triton X-100, 1 mM PMSF, 4 mM EDTA). Samples were immunoprecipitated with an anti-LRP6 antibody. The precipitates were subjected to SDS/PAGE (10% separating gel) and western blotting by using an anti-LRP6 antibody as a primary antibody. (B) Ectodomain recombinants of human LRP6 and mouse LRP5 at 17 lgmL1 and 50 lgmL1 puriﬁed bovine ﬁbrinogen were incubated with 0.03 lgmL1 VAP1 in PBS for 0, 1 and 3 h at 37 °C. Samples (12 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) Human ﬁbronectin at 15 lgmL1 was incubated with each dose of VAP1 in PBS for 1 h at 37 °C. Samples (12 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (D) The VAP1-cleavage point is shown by a black arrow in the schematic representation of LRP6. Fragment sequences of 140-kDa and 60-kDa bands of LRP6 detected by mass spectroscopic analysis are shown by a double underline and dashed underline, respectively. (E) A mouse ectodomain recombinant of LRP6 at 10 lgmL1 was incubated with 0.1 lgmL1 VAP1 in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining.

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 2. VAP1 cleaves LRP6 at the activation region. (A) HeLa cells were incubated with or without 0.4 lgmL1 VAP1 in serum-free medium for 2 h at 37 °C. The cells (1.5 9 107 cells) were harvested with a lysis buffer (25 mM Tris/HCl, pH 7.4, 300 mM NaCl, 1.5 mM MgCl2, 0.5% Triton X-100, 1 mM PMSF, 4 mM EDTA). Samples were immunoprecipitated with an anti-LRP6 antibody. The precipitates were subjected to SDS/PAGE (10% separating gel) and western blotting by using an anti-LRP6 antibody as a primary antibody. (B) Ectodomain recombinants of human LRP6 and mouse LRP5 at 17 lgmL1 and 50 lgmL1 puriﬁed bovine ﬁbrinogen were incubated with 0.03 lgmL1 VAP1 in PBS for 0, 1 and 3 h at 37 °C. Samples (12 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) Human ﬁbronectin at 15 lgmL1 was incubated with each dose of VAP1 in PBS for 1 h at 37 °C. Samples (12 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (D) The VAP1-cleavage point is shown by a black arrow in the schematic representation of LRP6. Fragment sequences of 140-kDa and 60-kDa bands of LRP6 detected by mass spectroscopic analysis are shown by a double underline and dashed underline, respectively. (E) A mouse ectodomain recombinant of LRP6 at 10 lgmL1 was incubated with 0.1 lgmL1 VAP1 in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining.

Article Snippet: Supernatants were incubated overnight with 2.5 lg anti-human LRP6 polyclonal goat antibody (R&D Sytems, Inc.) at 4 °C.

Techniques: Activation Assay, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Silver Staining, Recombinant

Fig. 3. VAP1 cleaves LRP5 at the same site as that of LRP6. (A,B) A mouse ectodomain recombinant of LRP5 at 26 lgmL1 was incubated with each dose of VAP1 (A) and VAP2 (B) in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) Mouse LRP5 ectodomain recombinant at 70 lgmL1 was incubated with 7 lgmL1 VAP1 in PBS for 1 h at 37 °C. Samples (60 lL) were subjected to SDS/PAGE (5–20% precast SDS/PAGE gel) and stained with Coomassie Brilliant Blue R250. (D) The VAP1-cleavage point is shown by a black arrow in the schematic representation of LRP5. The fragment sequences of the 140-kDa band of LRP5 detected by mass spectroscopic analysis is shown by a double underline. (E) Amino acid residues from cleavage site position P5 to P50 of LRP6 and LRP5 are aligned. The VAP1-cleavage point is shown by a black arrow.

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 3. VAP1 cleaves LRP5 at the same site as that of LRP6. (A,B) A mouse ectodomain recombinant of LRP5 at 26 lgmL1 was incubated with each dose of VAP1 (A) and VAP2 (B) in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) Mouse LRP5 ectodomain recombinant at 70 lgmL1 was incubated with 7 lgmL1 VAP1 in PBS for 1 h at 37 °C. Samples (60 lL) were subjected to SDS/PAGE (5–20% precast SDS/PAGE gel) and stained with Coomassie Brilliant Blue R250. (D) The VAP1-cleavage point is shown by a black arrow in the schematic representation of LRP5. The fragment sequences of the 140-kDa band of LRP5 detected by mass spectroscopic analysis is shown by a double underline. (E) Amino acid residues from cleavage site position P5 to P50 of LRP6 and LRP5 are aligned. The VAP1-cleavage point is shown by a black arrow.

Article Snippet: Supernatants were incubated overnight with 2.5 lg anti-human LRP6 polyclonal goat antibody (R&D Sytems, Inc.) at 4 °C.

Techniques: Recombinant, Incubation, SDS Page, Silver Staining, Staining

Fig. 4. Substrates and cleavage points of VAP1. (A) Substrates cleaved by VAP1 are indicated. The cleaved fragments were subjected to N-terminal sequence analysis. P5 to P50 amino acid residues of the cleavage points are shown. (B) Docking model of VAP1 and the cleavage site moiety of LRP6. The inset indicates the substrate-binding cleft. VAP1 is shown by a white space-ﬁlling model. The cleavage site moiety of LRP6 is shown by a red space- ﬁlling model (in the upper ﬁgure) and by a ball-and-stick model (in the lower ﬁgure). Zn2+ and Glu336A, both of which are involved in catalysis, and Leu363A are shown by grey spheres. S10 and S30

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 4. Substrates and cleavage points of VAP1. (A) Substrates cleaved by VAP1 are indicated. The cleaved fragments were subjected to N-terminal sequence analysis. P5 to P50 amino acid residues of the cleavage points are shown. (B) Docking model of VAP1 and the cleavage site moiety of LRP6. The inset indicates the substrate-binding cleft. VAP1 is shown by a white space-ﬁlling model. The cleavage site moiety of LRP6 is shown by a red space- ﬁlling model (in the upper ﬁgure) and by a ball-and-stick model (in the lower ﬁgure). Zn2+ and Glu336A, both of which are involved in catalysis, and Leu363A are shown by grey spheres. S10 and S30

Article Snippet: Supernatants were incubated overnight with 2.5 lg anti-human LRP6 polyclonal goat antibody (R&D Sytems, Inc.) at 4 °C.

Techniques: Sequencing, Binding Assay

Fig. 5. LRP6 cleavage by VAP1 is involved in disruption of cell–cell junctions and haemorrhage. (A,B) A human ectodomain recombinant of LRP6 at 30 lgmL1 was incubated with 30 ngmL1 VAP1 and with 2 mgmL1 LRP-cleavage site antibody or control antibody in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE and silver staining (A). Cleavage inhibition is shown by the 140-kDa fragment density score (B). The data (n = 5; error bars correspond to standard errors) were compared by Student’s t test. (C) HUVECs were incubated with 140 ngmL1 VAP1 and with 1.35 mgmL1 LRP6-cleavage site antibody or control antibody in a medium for 1 h at 37 °C. After being ﬁxed, the cells were stained with anti-VE-cadherin antibody. Arrows indicate the remaining membrane VE-cadherin. Scale bar, 50 lm. (D,E) VAP1 with LRP6 cleavage site antiserum or control serum was intradermally injected into mice bisymmetrically. One hour later, haemorrhagic plaques on the inner surface of the skin were observed (D). Scale bar, 10 mm. Densitometry scores of VAP1-induced haemorrhage with the antiserum or control serum in each individual were compared by paired t test (E).

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 5. LRP6 cleavage by VAP1 is involved in disruption of cell–cell junctions and haemorrhage. (A,B) A human ectodomain recombinant of LRP6 at 30 lgmL1 was incubated with 30 ngmL1 VAP1 and with 2 mgmL1 LRP-cleavage site antibody or control antibody in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE and silver staining (A). Cleavage inhibition is shown by the 140-kDa fragment density score (B). The data (n = 5; error bars correspond to standard errors) were compared by Student’s t test. (C) HUVECs were incubated with 140 ngmL1 VAP1 and with 1.35 mgmL1 LRP6-cleavage site antibody or control antibody in a medium for 1 h at 37 °C. After being ﬁxed, the cells were stained with anti-VE-cadherin antibody. Arrows indicate the remaining membrane VE-cadherin. Scale bar, 50 lm. (D,E) VAP1 with LRP6 cleavage site antiserum or control serum was intradermally injected into mice bisymmetrically. One hour later, haemorrhagic plaques on the inner surface of the skin were observed (D). Scale bar, 10 mm. Densitometry scores of VAP1-induced haemorrhage with the antiserum or control serum in each individual were compared by paired t test (E).

Article Snippet: Supernatants were incubated overnight with 2.5 lg anti-human LRP6 polyclonal goat antibody (R&D Sytems, Inc.) at 4 °C.

Techniques: Disruption, Recombinant, Incubation, Control, SDS Page, Silver Staining, Inhibition, Staining, Membrane, Injection

Fig. 6. VAP1-cleavage site and/or LDLa domains of LRP5/6 are deleted in many venom-resitent animals. Deleted regions of LRP6 (A) and LRP5 (B) in animals are shown in schematic representation. LRP5/6 of animals associated with tolerance to snake bite-induced haemorrhage are indicated by red boxes. LRP5/6 of animals with moderate resistance and animals with unknown sensitivity to snake bite- induced haemorrhage are shown by orange and green boxes, respectively. The presented data are from NCBI RefSeq (Table S1). Except for humans, mice and rats, the data are all predicted sequences from the genome of each animal. Amino acid sequences of the cleavage site and LDLa region are shown in Fig. 7. Regarding the predicted king cobra LRP6 (ETE69657.1), although the genome sequence of the full- length mRNA region of LRP6 contains several gaps, the indicated deleted region does not contain gaps.

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 6. VAP1-cleavage site and/or LDLa domains of LRP5/6 are deleted in many venom-resitent animals. Deleted regions of LRP6 (A) and LRP5 (B) in animals are shown in schematic representation. LRP5/6 of animals associated with tolerance to snake bite-induced haemorrhage are indicated by red boxes. LRP5/6 of animals with moderate resistance and animals with unknown sensitivity to snake bite- induced haemorrhage are shown by orange and green boxes, respectively. The presented data are from NCBI RefSeq (Table S1). Except for humans, mice and rats, the data are all predicted sequences from the genome of each animal. Amino acid sequences of the cleavage site and LDLa region are shown in Fig. 7. Regarding the predicted king cobra LRP6 (ETE69657.1), although the genome sequence of the full- length mRNA region of LRP6 contains several gaps, the indicated deleted region does not contain gaps.

Article Snippet: Supernatants were incubated overnight with 2.5 lg anti-human LRP6 polyclonal goat antibody (R&D Sytems, Inc.) at 4 °C.

Techniques: Combined Bisulfite Restriction Analysis Assay, Sequencing

Fig. 8. Recombinant human ADAM8 and ADAM12 cleave LRP6 at the same site as that of VAP1. (A) Recombinant human LRP6 at 64 lgmL1 was incubated with 20 lgmL1 recombinant human ADAM8 for 3 h at 37 °C. Samples (10 lL) were subjected to SDS/PAGE (12% separating gel) and silver staining. (B) LRP6 at 20 lgmL1 was incubated with 20 lgmL1 recombinant human ADAM12 for 16 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) LRP6 at 20 lgmL1 was incubated with 200 lgmL1 recombinant human ADAM12 for 18 h at 37 °C. Fragments containing C-terminal Fc peptide (20 lL) were puriﬁed with protein A–Sepharose. Samples were subjected to SDS/PAGE (15% separating gel) and silver staining. (D) The LRP6 140-kDa fragments treated with ADAM8 (A) and ADAM12 (B) and the LRP6 60-kDa fragment treated with ADAM12 (C) were analysed by mass spectrometry. Detected fragments by mass spectrometry and the proposed cleaved sites are shown.

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 8. Recombinant human ADAM8 and ADAM12 cleave LRP6 at the same site as that of VAP1. (A) Recombinant human LRP6 at 64 lgmL1 was incubated with 20 lgmL1 recombinant human ADAM8 for 3 h at 37 °C. Samples (10 lL) were subjected to SDS/PAGE (12% separating gel) and silver staining. (B) LRP6 at 20 lgmL1 was incubated with 20 lgmL1 recombinant human ADAM12 for 16 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) LRP6 at 20 lgmL1 was incubated with 200 lgmL1 recombinant human ADAM12 for 18 h at 37 °C. Fragments containing C-terminal Fc peptide (20 lL) were puriﬁed with protein A–Sepharose. Samples were subjected to SDS/PAGE (15% separating gel) and silver staining. (D) The LRP6 140-kDa fragments treated with ADAM8 (A) and ADAM12 (B) and the LRP6 60-kDa fragment treated with ADAM12 (C) were analysed by mass spectrometry. Detected fragments by mass spectrometry and the proposed cleaved sites are shown.

Article Snippet: Supernatants were incubated overnight with 2.5 lg anti-human LRP6 polyclonal goat antibody (R&D Sytems, Inc.) at 4 °C.

Techniques: Recombinant, Incubation, SDS Page, Silver Staining, Mass Spectrometry

Fig. 9. Hypothetical roles of ADAM and LRP5/6 in haemorrhage and invasion. (1) Cell–cell junctions with cadherin and catenin are normally stable. LRP5/6 may form a complex with cadherin and catenin. (2) Haemorrhagic SVMP, which is an ADAM-type toxin, can cleave LRP5/6 at the activation region. Cleaved LRP5/6 may form a dimer or multimer. (3) Cleaved LRP6 is involved in c-catenin and VE-cadherin relocation and in disruption of cell–cell adhesions. (4) Cleaved LRP6 mediates haemorrhage. Some animals with snake venom tolerance have deleted LRP5/6. (20) Invasion-associated ADAMs are located at the tips of invadopodia in invasive cells such as leukocytes and cancer cells. The ADAMs can cleave the same site of LRP6. (40) Cleaved LRP6 has the potential to cause disruption of cell–cell adhesions and to induce invasion. Summarizing the above, it is thought that ADAMs, as cell barrier openers, cleave novel ADAM receptors, LRP5/6, to induce haemorrhage and potentially invasion.

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 9. Hypothetical roles of ADAM and LRP5/6 in haemorrhage and invasion. (1) Cell–cell junctions with cadherin and catenin are normally stable. LRP5/6 may form a complex with cadherin and catenin. (2) Haemorrhagic SVMP, which is an ADAM-type toxin, can cleave LRP5/6 at the activation region. Cleaved LRP5/6 may form a dimer or multimer. (3) Cleaved LRP6 is involved in c-catenin and VE-cadherin relocation and in disruption of cell–cell adhesions. (4) Cleaved LRP6 mediates haemorrhage. Some animals with snake venom tolerance have deleted LRP5/6. (20) Invasion-associated ADAMs are located at the tips of invadopodia in invasive cells such as leukocytes and cancer cells. The ADAMs can cleave the same site of LRP6. (40) Cleaved LRP6 has the potential to cause disruption of cell–cell adhesions and to induce invasion. Summarizing the above, it is thought that ADAMs, as cell barrier openers, cleave novel ADAM receptors, LRP5/6, to induce haemorrhage and potentially invasion.

Article Snippet: Supernatants were incubated overnight with 2.5 lg anti-human LRP6 polyclonal goat antibody (R&D Sytems, Inc.) at 4 °C.

Techniques: Activation Assay, Disruption

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