Fig. 2.
ApoE4 disrupts Reelin-induced enhancement of NMDA receptor-mediated Ca2+ influx, phosphorylation, and glutamate receptor trafficking. Representative traces of fura-2 340/380 ratios recorded from neurons preincubated with media containing (filled circles) or not containing (open circles) 5 nM Reelin and no ApoE (A), or 3 μg/mL ApoE2 (B), ApoE3 (C), or ApoE4 (D). Response delay due to wash-in of glutamate is ≈20 s. (E) Averaged ΔR (the maximal difference in 340/380 ratio induced by glutamate) recorded from primary neurons subjected to the indicated treatments in the absence or presence of ∼5 nM Reelin and 0, 1, or 3 μg/mL ApoE2, -E3, or -E4 (n = 20–50). Asterisks above individual columns indicate significance (P < 0.001) compared with control neurons receiving no treatment (ApoE nor Reelin). Asterisks on brackets indicate significant difference (P < 0.001) between treatments; unpaired t test. (F) Native but not boiled (CR50*) function blocking CR50 antibody prevents Reelin-dependent Ca2+ influx. (G) ApoE4 diminishes Reelin-induced GluN2A and B tyrosine phosphorylation (pGluN2 compared with total cellular GluN2). β-actin, loading control. (H and I) ApoE4 reduces GluA1, GluA2/3, and GluN2B cell-surface levels in acutely Reelin stimulated primary neurons. GluN2A surface levels were too low for reliable detection.