Figure 5.

In vitro treatments with 1α, 25-dihydroxivitamin D3 or dexamethasone diminish SM amount, increase NSM protein levels and activity, and restore RhoA membrane binding and filamentous actin levels in ASMko synapses.

1. Western blot of NSM protein levels in total (Tot) and synaptosomal (Syn) fractions from wt and ASMko mice brains containing the same amount of protein.
2. Mean ± s.d. of SM levels (nmol/mg protein) in ASMko synaptosomes treated or not with 1α, 25-dihydroxivitamin D3 (VitD3) or dexamethasone (DM) (n = 3, *P_vitD3 = 0.04; *P_DM=0.033).
3. Western blot of NSM and tubulin levels in ASMko synaptosomes treated or not with VitD3 and dexamethasone. Graph shows mean ± s.d. of NSM protein levels normalized to tubulin (n = 3, P = 0.025). Graphs to the right show mean ± s.d. of NSM activity in ASMko synaptosomes treated or not with dexamethasone (n = 3, *P = 0.032).
4. Western blots of RhoA levels in supernatants (S) and pellets (P) after 100,000 g centrifugation of ASMko synaptosomes treated or not with VitD3 or DM. Graph shows mean ± s.d. of the RhoA ratio pellet/supernatant in the treated samples as percentage of ASMko non treated samples that were considered 100% (n = 3; *P_vitD3 = 0.042; **P_DM = 0.001).
5. Top: Dendrites from ASMko neurons non treated or treated with vitaminD3 or dexamethasone stained for MAP2 (blue), PSD95 (red), and phalloidin (green); bottom: phalloidin staining only. The graph shows mean ± s.d. of phalloidin fluorescence intensity per spine area (n = 250 dendritic spines from 3 independent cultures, **P_vitD3 = 0.001; ***P_DM = 0.0008). Bars: 5 μm.