Soluble Aβ oligomers isolated by SEC from human postmortem AD brain induces rapid CaN activation in spines, alters spine morphology, and reduces spine F-actin and synaptic surface GluR1. A, B, Quantitative analysis of normalized spectral ratio (intensity ratio of 565–522 nm) from different neuron compartments in neurons exposed to control or AD SEC fractions with or without 3D6 or FK506. Neurons treated with AD SEC fractions for 60 min show significantly higher FRET of both CaNA–CaM (A) and CaNA–CaNB (B) in spines compared with neurons treated with the comparable SEC fraction from a control brain, whereas FRET signal of CaNA–CaM or CaNA–CaNB in the cytosolic and nuclear compartments was not changed in neurons treated with either control or AD SEC fractions (n ≥ 45 cells and n ≥ 200 spines; **p < 0.001). Data represent mean ± SEM. C, Quantification of filopodia-like protrusions (percentage of total spines). AD SEC fraction exposure for a period of 60 min caused spine length elongation and induced more filopodia-like protrusions. This response was completely inhibited when cells were pretreated with 1 μm FK506 or Tg2576-containing conditioned media was immunodepleted with 3D6. D, Exposure of neurons to oligomeric Aβ-enriched SEC fractions prepared from AD brain samples for 60 min caused no changes in spine density compared with that of neurons exposed to similar SEC fractions prepared from control brain. E, F, Decreases in spine F-actin and synaptic surface GluR1 subunit of AMPA receptors during rapid CaN activation induced by SEC-isolated Aβ oligomers prepared from AD human brains. Neurons treated with AD SEC fraction for 60 min at indicated experimental conditions (with or without 3D6 immunodepletion or in the presence or absence of the CaN inhibitor FK506), and then stained with an antibody to the extracellular N-terminal domain of GluR1 to label surface AMPA receptors, and phalloidin to visualize F-actin (E). Exposure of neurons to SEC-isolated Aβ oligomers prepared from AD human brains reduces the ratios of both phalloidin-stained F-actin and surface GluR1 on the spine to the dendritic shafts, indicating a loss of F-actin and synaptic GluR1 from dendritic spines. This effect was blocked by 3D6 and FK506 (F). n = total 200 spines in each condition, from three independent experiments. **p < 0.001. Data are presented as mean ± SEM.