PrPCaccumulates in intracellular compartments in ADAM10 cKO neurons. (A) Confocal (upper two rows; scale bar overview: 100 μm, scale bar single neuron: 10 μm) and spinning disc (lower two rows; scale bar YZ-sections: 10 μm) immunofluorescence microscopy for PrPC in permeabilized wildtype neurons (Ctrl) shows PrPC in evenly distributed vesicular structures (open arrowhead). In ADAM10 cKO neurons, dense PrPC accumulations are detectable adjacent to the nucleus (filled arrowhead) and in neuronal processes. YZ-sections through individual neurons confirm these findings (arrow for PrPC accumulation in ADAM10 cKO). Asterisks mark nuclei. (B) Confocal microscopic analysis of co-stainings of PrPC (left column) with intracellular organelle marker proteins (middle column) in wildtype littermate (Control) and ADAM10 cKO neurons. Overlays are shown in the right column. Accumulations of PrPC are only found in ADAM10 cKO neurons (filled arrowheads in d, j, and p) and colocalize with protein disulfide isomerase (PDI) used as marker for ER (e, f) and Golgi marker GM130 (k, l). Partial colocalization of PrPC with the lysosomal marker LAMP1 is detectable in vesicular structures (q, r). Scale bars represent 10 μm. Asterisks in overlays mark nuclei.