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anti lrp6 af647 (R&D Systems)

Name: anti lrp6 af647
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Rating: 93 (3 reviews)

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R&D Systems anti lrp6 af647
Anti Lrp6 Af647, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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( A ) Co-staining of iPSC-derived cortical neurons (DIV60) to observe Wnt-7a and LRP6 localisation. Wnt-7a can be seen localised on dendritic protrusions (yellow outlines), whereas LRP6 generally localises at apposing membranes (orange outlines). Wnt-7a-positive protrusions also harbour proteins associated with Wnt-signalling filopodia, such as Flotillin-2 (Flot-2; B ; yellow outlines) and Wntless/evenness interrupted (Wls’; C ; yellow outlines), whereas Wnt-signalling filopodia marker Myo-10 localise to a Wnt-7a-negative subset of protrusions ( D ; orange outlines). Flot-2 and Wls also co-colocalise on the filopodia ( E ; yellow outlines). ( F ) Quantification identified a subset of Wnt-7a-positive protrusions, with a significant difference observed in the length of Wnt-7a-positive filopodia, compared to Wnt-7a-negative filopodia. ( G ) Quantification of co-clustering of LRP6 and Wnt-7a at apposing membranes identified a significant increase in co-clustering of Wnt-7a-positive mushroom-shaped protrusions and LRP6 when compared to co-clustering at filopodia contacts. ( H ) Quantification of protrusion number based on cytoneme markers found significantly more Wnt-7a/Flot-2-positive and Wnt-7a/Wls-positive protrusions when compared to Wnt-7a-only positive protrusions, and over 75% of all protrusion analysed were Wnt-7a positive ( I & J ). ( K ) Quantification of protrusion length found no difference based on cytoneme protein expression ( K ). The co-clustering of Wnt-7a-positive protrusions and apposed LRP6 ( L ) was dependent on both palmitoleation ( O ) and calcium signals ( R ). Furthermore, colocalisation of Wnt-7a with the post-synaptic protein PSD95 ( M, P, S ) and the pre-synaptic protein Bassoon (BSN; N, Q, T ), was also dependent on palmitoleation and calcium signalling, as quantified in ( U ). Statistical significance was addressed using one-way ANOVA with Dunnett’s multiple comparison test to compare relevant controls within groups. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

Journal: bioRxiv

Article Title: Wnt-7a-positive dendritic cytonemes induce synaptogenesis in cortical neurons

doi: 10.1101/2023.02.17.528927

Figure Lengend Snippet: ( A ) Co-staining of iPSC-derived cortical neurons (DIV60) to observe Wnt-7a and LRP6 localisation. Wnt-7a can be seen localised on dendritic protrusions (yellow outlines), whereas LRP6 generally localises at apposing membranes (orange outlines). Wnt-7a-positive protrusions also harbour proteins associated with Wnt-signalling filopodia, such as Flotillin-2 (Flot-2; B ; yellow outlines) and Wntless/evenness interrupted (Wls’; C ; yellow outlines), whereas Wnt-signalling filopodia marker Myo-10 localise to a Wnt-7a-negative subset of protrusions ( D ; orange outlines). Flot-2 and Wls also co-colocalise on the filopodia ( E ; yellow outlines). ( F ) Quantification identified a subset of Wnt-7a-positive protrusions, with a significant difference observed in the length of Wnt-7a-positive filopodia, compared to Wnt-7a-negative filopodia. ( G ) Quantification of co-clustering of LRP6 and Wnt-7a at apposing membranes identified a significant increase in co-clustering of Wnt-7a-positive mushroom-shaped protrusions and LRP6 when compared to co-clustering at filopodia contacts. ( H ) Quantification of protrusion number based on cytoneme markers found significantly more Wnt-7a/Flot-2-positive and Wnt-7a/Wls-positive protrusions when compared to Wnt-7a-only positive protrusions, and over 75% of all protrusion analysed were Wnt-7a positive ( I & J ). ( K ) Quantification of protrusion length found no difference based on cytoneme protein expression ( K ). The co-clustering of Wnt-7a-positive protrusions and apposed LRP6 ( L ) was dependent on both palmitoleation ( O ) and calcium signals ( R ). Furthermore, colocalisation of Wnt-7a with the post-synaptic protein PSD95 ( M, P, S ) and the pre-synaptic protein Bassoon (BSN; N, Q, T ), was also dependent on palmitoleation and calcium signalling, as quantified in ( U ). Statistical significance was addressed using one-way ANOVA with Dunnett’s multiple comparison test to compare relevant controls within groups. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

Article Snippet: The following primary antibodies were used for immunofluorescence: anti-Wnt7a (abcam; ab100792), anti-LRP6 (BioTechne; FAB1505R), anti-Bassoon (abcam; ab82958), anti-PSD95 (abcam; ab18258), anti-PSD95 (abcam; ab13552), anti-Flotillin-2 (Santa Cruz; sc-28320), anti-Flotillin-2 (abcam; ab113661), anti-Myosin-X (Santa Cruz C-1; sc-166720), anti-Evi (EMD Milipore; YJ5).

Techniques: Staining, Derivative Assay, Marker, Expressing, Comparison

( A ) Schematic representation of the tethering experiment using double transfection of Wnt-7a-GFP and the Morphotrap nanobody construct, Vhh-CD8-mCh that binds GFP-tagged proteins. ( B ) Colocalisation of mem-mCh and Wnt-7a-GFP on protrusions. ( C ) Significantly higher Wnt-7a-GFP colocalisation on filopodia is observed when co-transfected with Morphotrap, quantified by Pearson’s correlation coefficient (PCC; D ). ( E, E’ ) Transfection of iPSC-derived cortical neurons with mem-GFP and mem-mCh, followed by post-staining for LRP6 and the actin cytoskeleton (Phalloidin). ( F, F’ ) Transfection of iPSC-derived cortical neurons with Wnt-7a-GFP and mem-mCh, followed by post-staining for LRP6 and the actin cytoskeleton (Phalloidin) identified Wnt-7a-GFP positive protrusions that cluster LRP6 in apposed cells. ( G, G’ ) Transfection with Wnt-7a-GFP and Morphotrap, followed by post-staining for LRP6 and actin, identified protrusions harbouring membrane-tethered Wnt-7a-GFP that cluster LRP6 in apposed cells. ( H ) Quantification of co-clustering of LRP6 with Wnt-7a-GFP. ( I, I’ ) Transfection of iPSC-derived cortical neurons with mem-GFP and mem-mCh, followed by post-staining for PSD95 and the actin cytoskeleton (Phalloidin). ( J, J’ ) Transfection with Wnt-7a-GFP and mem-mCh, followed by post-staining for PSD95 and actin, found co-localisation on dendritic protrusions. ( K, K’ ) Wnt-7a-GFP co-transfected with Morphotrap and co-localised with PSD95 on dendritic protrusions. ( L ) Quantification of co-localisation of PSD95 with Wnt-7a-GFP +/− morphotrap on dendritic protrusions. Statistical significance was addressed using One way ANOVA with Dunnett’s post hoc test for multiple comparisons, comparing groups to the mem-mCh/mem-GFP control group.

Journal: bioRxiv

Article Title: Wnt-7a-positive dendritic cytonemes induce synaptogenesis in cortical neurons

doi: 10.1101/2023.02.17.528927

Figure Lengend Snippet: ( A ) Schematic representation of the tethering experiment using double transfection of Wnt-7a-GFP and the Morphotrap nanobody construct, Vhh-CD8-mCh that binds GFP-tagged proteins. ( B ) Colocalisation of mem-mCh and Wnt-7a-GFP on protrusions. ( C ) Significantly higher Wnt-7a-GFP colocalisation on filopodia is observed when co-transfected with Morphotrap, quantified by Pearson’s correlation coefficient (PCC; D ). ( E, E’ ) Transfection of iPSC-derived cortical neurons with mem-GFP and mem-mCh, followed by post-staining for LRP6 and the actin cytoskeleton (Phalloidin). ( F, F’ ) Transfection of iPSC-derived cortical neurons with Wnt-7a-GFP and mem-mCh, followed by post-staining for LRP6 and the actin cytoskeleton (Phalloidin) identified Wnt-7a-GFP positive protrusions that cluster LRP6 in apposed cells. ( G, G’ ) Transfection with Wnt-7a-GFP and Morphotrap, followed by post-staining for LRP6 and actin, identified protrusions harbouring membrane-tethered Wnt-7a-GFP that cluster LRP6 in apposed cells. ( H ) Quantification of co-clustering of LRP6 with Wnt-7a-GFP. ( I, I’ ) Transfection of iPSC-derived cortical neurons with mem-GFP and mem-mCh, followed by post-staining for PSD95 and the actin cytoskeleton (Phalloidin). ( J, J’ ) Transfection with Wnt-7a-GFP and mem-mCh, followed by post-staining for PSD95 and actin, found co-localisation on dendritic protrusions. ( K, K’ ) Wnt-7a-GFP co-transfected with Morphotrap and co-localised with PSD95 on dendritic protrusions. ( L ) Quantification of co-localisation of PSD95 with Wnt-7a-GFP +/− morphotrap on dendritic protrusions. Statistical significance was addressed using One way ANOVA with Dunnett’s post hoc test for multiple comparisons, comparing groups to the mem-mCh/mem-GFP control group.

Techniques: Transfection, Construct, Derivative Assay, Staining, Membrane

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.

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