A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer’s disease

Synapse loss strongly correlates with cognitive decline in Alzheimer’s disease (AD), but the underlying mechanisms are poorly understood. Deficient Wnt signaling contributes to synapse dysfunction and loss in AD. Consistently, a variant of the LRP6 receptor, (LRP6-Val), with reduced Wnt signaling, is linked to late-onset AD. However, the impact of LRP6-Val on the healthy and AD brain has not been examined. Knock-in mice, generated by gene editing, carrying this Lrp6 variant develop normally. However, neurons from Lrp6-val mice do not respond to Wnt7a, a ligand that promotes synaptic assembly through the Frizzled-5 receptor. Wnt7a stimulates the formation of the low-density lipoprotein receptor-related protein 6 (LRP6)–Frizzled-5 complex but not if LRP6-Val is present. Lrp6-val mice exhibit structural and functional synaptic defects that become pronounced with age. Lrp6-val mice present exacerbated synapse loss around plaques when crossed to the NL-G-F AD model. Our findings uncover a previously unidentified role for Lrp6-val in synapse vulnerability during aging and AD.


Figure S2 .
Figure S2.Characterisation of homozygous Lrp6-val knock-in mice.A) Images of WT and Lrp6-val mice at 7 months showed that these mice developed normally and have no visible external abnormalities.B) Adult Lrp6-val mice had the same weights as control WT mice.Weights of male mice were measured at 4-8 months of age.WT N = 9.Lrp6-val N = 10.Unpaired T-test.C) Quantitative RT-PCR analyses of Lrp6 mRNA levels in the hippocampus of WT and Lrp6-val mice at 3-4 months of age showed no changes in the expression of Lrp6.WT N = 5, Lrp6-val N = 6.Unpaired T-Test.D) Hippocampal LRP6 protein levels in WT and Lrp6-val mice at 4 months.E) No changes in LRP6 levels (normalised to β-actin) were observed between WT or Lrp6-val mice.N = 3 per genotype.Unpaired T-test.F) SIM images of excitatory synapses containing LRP6 (red) from WT and Lrp6-val hippocampal neurons showing vGlut1 (blue) and PSD-95 (green).Scale bar = 0.2 μm.G) LRP6 receptor exhibited similar pre-and post-synaptic localisation in WT and Lrp6-val mice. 2 independent cultures, 6-8 images per culture.Unpaired T-tests.Data are represented as mean  SEM.

Figure S3 .
Figure S3.Basal synaptic transmission is unaffected in Lrp6-val mice at 7-9 months and vesicle recycling and initial fusion efficiency are unchanged in Lrp6-val mice at 12-14 months.A) Representative traces of post-synaptic currents elicited at different stimulation intensities.B) No differences were detected in the input-output curves at 7-9 months.N = 12-14 cells recorded from 4-5 animals per genotype.Repeated-measures one-way-ANOVA.C) Graphs display the recycling rate and initial fusion efficiency obtained from all cells.No differences were observed in Lrp6-val mice when compared to WT mice at 12-14 months.N = 12 cells from 4 animals per genotype.Unpaired Student's T-test.Data are represented as mean  SEM.

Figure S4 .
Figure S4.Lrp6-val mice do not display neuronal loss at 16-18 months.A) Confocal images of the hippocampus of WT and Lrp6-val mice labelled with DAPI (blue) and NeuN (red).Scale bar = 150 μm.Insets show higher magnification images of the CA1 region.Scale bar = 100 μm.B) Quantification revealed no differences in the percentage of NeuN positive cells between WT and Lrp6-val mice.WT N = 6, Lrp6-val N = 5.Unpaired T-test.Data are represented as mean  SEM.

Figure S7 .
Figure S7.Lrp6-val neurons do not respond to Wnt7a or Wnt3a to promote synapse formation.A) Diagram showing the isolation of primary neurons from WT and homozygous Lrp6-val mice to evaluate synapse number or surface levels of the receptor by surface biotinylation.Top: neurons were exposed to recombinant Wnt7a or Wnt3a for 3 hours prior to analyses of synapses by confocal microscopy.Bottom: neurons were incubated with biotin (red circles).Biotin bound surface proteins (Green and blue shapes) were pulled down with streptavidin-agarose beads (blue circles).B) Wnt7a (100ng/ml) increased the number of synapses in WT neurons, but Wnt7a had no effect on Lrp6-val neurons.N = 3 independent cultures, 8-11 images per culture.Two-way-ANOVA with Games-Howell post hoc test.* p < 0.05.C) Wnt3a increased synapse number in WT neurons but Lrp6-val neurons failed to respond to Wnt3a.N = 3 independent cultures, 8-10 images per culture.Two-way-ANOVA with Games-Howell post hoc test.* p < 0.05.D) Surface biotinylation analyses of LRP6 were performed on neurons isolated from WT and homozygous Lrp6-val mice.E) No differences in the ratio of surface to total LRP6 were observed.N = 3 independent cultures.Mann-Whitney.Data are represented as mean  SEM.

Figure S8 .
Figure S8.The surface localisation of LRP6 is not affected in cells expressing LRP6-Val.A) Schematic of surface biotinylation analyses of HeLa cells.B) Western blot analyses of LRP6 levels following surface biotinylation of cells expressing Fz5-HA and WT LRP6 or LRP6-Val and treated with recombinant Wnt7a.C) No differences in the ratio of surface to total LRP6 were observed after Wnt7a treatment.N = 3 independent cultures.Two-way-ANOVA with Games-Howell post hoc test.D) Western blot analyses of Fz5-HA following surface biotinylation of HeLa cells expressing Fz5-HA and WT LRP6 or LRP6-Val and treated with recombinant Wnt7a.We observed a higher molecular weight of Fz5-HA at the surface, which is probably due to changes in glycosylation of this receptor (59).E) The ratio of surface to total Fz5-HA were unchanged after Wnt7a treatment.Both bands observed for HA were quantified.N = 3 independent cultures.Twoway-ANOVA with Games-Howell post hoc test.F) Confocal images of HeLa cells expressing GFP, WT LRP6 and Fz5-HA or GFP, LRP6-Val and Fz5-HA.Total LRP6 (red) and phosphorylated LRP6 (pLRP6) (grey).Scale bar = 2 μm.Data are represented as mean  SEM.

Figure S11 .
Figure S11.Lrp6-val does not affect synapse number in NL-G-F mice at 2 months or 10 months.A) Confocal images of the CA1 SR of WT, Lrp6-val, NL-G-F and NL-G-F;Lrp6-val mice at 2 months showing Bassoon (green) and Homer1 (red) puncta.Scale bar = 3.8 μm.Insets show higher magnification images of synapses.Scale bar = 2 μm B) Quantification of Bassoon and Homer1 puncta and synapse number (co-localised puncta).No differences are detected between any of the different genotypes.WT N = 5, Lrp6-val N = 4, NL-G-F N = 5, NL-G-F; Lrp6-val N = 6.One-way-ANOVA with Tukey's post hoc test.C) Confocal images of synapses, co-localised Bassoon (green) and Homer1 (red) puncta, at increasing distances from the centre of an Aβ plaque (blue) in NL-G-F and NL-G-F;Lrp6-val mice at 10 months.D) Quantification revealed no differences in synapse number.Scale bar = 5 μm.NL-G-F N = 15 slices from 5 brains, NL-G-F; Lrp6-val N = 11 slices from 4 brains.Repeated measure Two-way-ANOVA with Bonferroni's post hoc test.Data are represented as mean  SEM.