An sSORLA fragment enhances neurite outgrowth. A, Conditioned media from cultured WT or SORLA TG cortical neurons were sampled from DIV3 to DIV10 and immunoblotted for sSORLA or sAPPα as indicated. Neuronal cell lysates were also immunoblotted for full-length SORLA or APP as indicated (right). Relative sSORLA levels were quantified by densitometry (bottom, n = 3 experiments). sSORLA levels from DIV3 neurons were set to 1.0 (F_(3,16) = 5.307, DIV7 WT vs TG, p = 0.0036; DIV10 WT vs TG, p < 0.0001, two way ANOVA with Tukey's multiple comparisons). B, sSORLA (sSORLA-FLAGhis₆) expressed in HEK293T cells was purified from conditioned medium using nickel NTA-agarose, and subjected to Ponceau S staining following transfer onto nitrocellulose membranes. BSA was stained to determine the concentration of purified sSORLA. C, WT hippocampal neurons at DIV2 were incubated overnight with 150 ng/ml purified sSORLA, and stained for tubulin (green), F-actin (red), and DAPI. Scale bar, 10 µm. Graphs represent quantification of the length of all neurites in WT control (black) and sSORLA (blue) treated hippocampal neurons (top) or the neurite length averaged per neuron (bottom) (control, 164 neurites, 46 neurons; sSORLA, 182 neurites, 55 neurons; 6 coverslips, 3 independent dissections/cultures per treatment; top: p < 0.0001; bottom: p = 0.0027, t test). D, Axon length was measured in hippocampal neurons identified with axonal extensions in control and sSORLA-treated WT neurons (control, 30 axons/neurons; sSORLA, 48 axons/neurons; 6 coverslips, 3 independent dissections/cultures per treatment) (p = 0.0008, t test). E, The number of neurites was scored in control and sSORLA-treated WT hippocampal neurons (control, 164 neurites scored in 46 neurons; sSORLA, 182 neurites scored in 55 neurons; 6 coverslips, 3 independent dissections/cultures per treatment). A, E, Data are mean ± SE. C, D, Data are mean ± SD. C, D, **p < 0.01; ***p < 0.001; Student's t test; A, Two-way ANOVA followed by Tukey's multiple comparisons.