Fig. 2: Cholesterol alterations induce mGluR5 changes upon NPC1 deficiency.
From: Enhanced mGluR5 intracellular activity causes psychiatric alterations in Niemann Pick type C disease
A Image of BODIPY-cholesterol and Lamp1 staining in cultured neurons from wt mice treated with Veh, U18 alone or with U18 plus MCD. Graph shows mean ± SEM BODIPY-cholesterol fluorescence intensity (a.u.) in Lamp1 positive area compared to cytosol (P Veh-U18 = 0.0383; P U18-U18+MCD = 0.0290; n = 3; Paired two-tailed t test). Scale bar 5 µm. B–E IFA of mGluR5 in cultured neurons from wt mice treated with Veh, U18 alone or with U18 plus MCD. B mGluR5 total staining in permeabilized neurons. C mGluR5 surface staining using anti-mGluR5 N-terminal in non -permeabilized neurons. D mGluR5 co-staining with Lamp1. E mGluR5 co-staining with Homer 1. Graphs in (B, C) show mean ± SEM mGluR5 fluorescence intensity as a percentage of the levels in the neurons treated with Veh that were considered 100% (PB. Veh-U18 = 0.0243; PB. U18-U18+MCD = 0.0378; PC. Veh-U18 = 0.0471; n = 4–6; Grouped one-way ANOVA Bonferroni post hoc). Scale bar 10 µm. Graphs in (D, E) show mean ± SEM Manders’ coefficient of co-localization between mGluR5 and Lamp1 or Homer1 (PD. Veh-U18 = 0.0094; PD. U18-U18+MCD = 0.0314; PE. Veh-U18 = 0.0419; n = 4–6; Grouped one-way ANOVA Bonferroni post hoc). Scale bar 5 µm. F IFA of mGluR5 co-stained with the raft marker caveolin1 in cultured neurons from wt mice treated with Veh or with U18. Graphs show mean ± SEM Manders’ coefficient of co-localization between mGluR5 and caveolin1 (n = 4). Scale 10 µm. G Western blot showing mGluR5 in the supernatant (detergent-resistant membrane; DRM) or pellet (non DRM) obtained after cold detergent extraction in cultured neurons from wt mice treated with U18 or Veh. The DRM canonical marker flotilin was used as control for the isolation protocol. Graph shows mean ± SEM levels of mGluR5 expressed as a percentage of the total levels (n = 6; Two-way ANOVA Bonferroni post hoc).
