Figure 1

Loss of APP and APPs-α application each increase neurite length. E18 primary hippocampal or cortical neurons from wild-type (WT) or APP knock-out (APP KO) mice were treated as described. Three days later, the cells were fixed and immunostained for βIII-tubulin, and neurite length was measured. (a) Comparison of neurite lengths of wild type and APP knock-out hippocampal neurons. (b) Comparison of neurite lengths of wild-type hippocampal neurons treated with CM from untransfected, APPs, APLP1, or APLP2 expressing CHO cells. (c) Western blots for APPs (8E5) on CM from day 0 (d0), d1, or d2 after addition to primary neurons (left panel) or western blot of equal amounts of purified APPs-α compared to APPs-α conditioned media (right panel). (d) Silver stain of sequential purification steps of APPs-α from baculovirus-transduced insect cells. Left panel: lanes 1 and 2 are ammonium sulfate (A.S.) precipitations from 0–60% or 60–100%; lane 3 is the flow through (FT) after addition to a nickel chelating column; lanes 4–6 are washes from the column; and lanes 7–9 are elutes from column. Right panel: final purified product of APPs-α with mutation of cysteine 117 (see Materials and methods for details of purification). Bottom: western blot (WB) with an antibody that recognizes the extracellular domain of APP (8E5, Elan) (e) Quantification of neurite length after addition of purified APPs-α (wild type) at increasing concentrations or APPs-α (C117A) at 120 ng/ml. (f) E18 primary cortical neurons from APP knock-out or wild-type littermates were treated with CM from either CHO cells stably expressing APPs-α or control CHO cells. Three days later, cells were fixed and immunostained for βIII-tubulin, and neurite length was measured. Error bars represent standard error of the mean; *p < 0.05; ***p < 0.001.