Figure 6.
Mixed-culture analysis of APP synaptogenic activity.
A
, Representative images of HEK293 cells transfected with GFP (GFP), neuroligin/GFP (NL), or APP/GFP (APP) (each at 10:1 construct/GFP molar ratio), cocultured with wild-type hippocampal neurons, and stained with anti-synaptophysin (Syn) and anti-MAP2 (Map2) antibodies. Syn-positive, but Map2-negative puncta could be seen atop of NL and APP transfected HEK293 cells (marked in green), but not GFP vector control. The images are displayed either individually or merged (last column, GFP, green; Syn, red; Map2, blue). Scale bar, 20 μm.
B
, Quantification of the average area of HEK293 cells covered by synaptophysin immunoreactivity. Error bars indicate SEM.
C
, Quantification of average number of Syn-positive puncta per transfected HEK293 cell. **p < 0.01; ***p < 0.001 (t test).
D
, Double immunostaining of APP transfected HEK293/hippocampal cocultures with anti-APP and anti-SV2 antibodies. The images are displayed either individually or merged (SV2, red; APP, green). Selected SV2 and APP double-positive punctas are marked by arrowheads. Scale bar, 20 μm.
E
, Introduction of a Flag-tagged APP in neurons and coculture with APP-expressing HEK293 cells (APP/GFP). APP-expressing axon is marked by arrow. SV2, Immunostaining with the anti-SV2 antibody to identify the synaptic puncta atop HEK293 cells; Flag, immunostaining with the anti-Flag antibody to recognize neuronal expressed APP. SV2- and Flag-positive punctas are highlighted by arrowheads. Merge, Overlap of the three panels (293 cells, green; SV2, red; Flag, blue with overlap of the three in white).
F
, Schematic diagram of the APP constructs. Shaded rectangle at the N terminus of each construct represents the APP signal peptide; E1 and E2, the E1 (amino acids 22-189) and E2 (amino acids 289–500) domains of the APP extracellular sequences; shaded square, APP transmembrane domain; shaded oval, GPI anchor.
G
, Western blot analysis of APP expression in transfected HEK293 cells using the 22C11 and APPc antibodies, which recognize residues within the E1 domain and the C-terminal end of APP respectively. Tubulin was used as a loading control.
H
, Cell surface APP expression assayed by biotinylation experiment.
I
,
J
, Quantification of the average area of HEK293 cells covered by synaptophysin immunoreactivity (
I
) and the average number of Syn-positive puncta per transfected HEK293 cell (
J
) induced by APP and its derivatives. The asterisks above each bar are in comparison with the full-length APP. The asterisks on top of the brackets are comparison between the specific pair. *p < 0.05; **p < 0.01; ***p < 0.001 (t test).