Fig. 6

GCS inhibition also protects primary hippocampal neurons upon ADDL exposure. a A TLC shows that a concentration of 1 μM GENZ (3 days) efficiently inhibits ganglioside biosynthesis in primary mouse hippocampal neurons. b Exposure to ADDLs decreases the viability of neurons, as determined by an MTT assay (white bar). However, GENZ treatment of primary neurons maintains viability upon ADDL stress (grey bar; 1 μM ADDLs, 24 h; n = 12 replicates). c Surface immunofluorescence of dendritic IR and ADDLs (antibody 6E10) on a non-permeabilized primary neuron (1 μM ADDLs, 30 min). The fluorescence intensity of IR staining has been measured along the dendrites (n = 169–285 measurements). ADDL exposure leads to IR removal from the dendrite, whereas dendritic IR are increased in GENZ-treated neurons. d Combined PLA/phalloidin staining shows the respective PLA complexes (green labels) on primary dendrites. IR interact with ADDLs and gangliosides GD1a and GT1b. ADDLs themselves are in close proximity of GD1a and GT1b. e Triple immunofluorescence directly visualizes the proposed IR/ADDL/GD1a complexes (white arrowheads). Areas with strong ADDL binding co-label with GD1a, but IR staining is weak due to IR removal (big green arrowheads). However, IR/ADDL/GD1a complexes are found at locations with weaker ADDL staining (small arrowheads). Unpaired two-tailed student’s t-test (if p ≤ 0.05, p ≤ 0.01, or p ≤ 0.001 results are marked with (*), (**) or (***), respectively); 1 μM ADDLs, 24 h. Scale bars: 5 μM