Fig. 1.

Fibrillar Aβ induces neuronal dystrophy, Tyr phosphorylation, and paxillin activation in cortical neurons. Aβ was added at day 5, and the cultures were processed at day 7 in culture.A, Neurites appear smooth and healthy incontrol cultures. Fibrillar Aβ-induced aberrant neurite morphology, including decreased caliber and acute angles and loops (Aβ, arrows). Neurons were immunolabeled with anti-tubulin class III antibody. Scale bar, 10 μm.B, Western blot of whole-cell homogenates developed with anti-phospho-Tyr antibody 4G10. Note the increase in phospho-Tyr in several bands after Aβ treatment (arrowheads) and decreased Tyr phosphorylation in a band of ∼180 kDa (small arrow). Con, Control. C, Homogenates were immunoprecipitated (IP) with anti-paxillin (α-pax) antibody and immunostained with 4G10. Note the increase in paxillin Tyr phosphorylation in Aβ-treated neurons (Aβ). Immunoprecipitates with nonimmune (NI) serum were negative. Con, Control; WB, Western blot. D, Paxillin Tyr phosphorylation was quantified by densitometry in vehicle-treated samples (Con) and normalized as 100%. Note the significant increase (170 ± 30%) induced by fibrillar Aβ (Aβ). The membranes were reprobed for paxillin to confirm equal loading. Values are mean ± SEM;n = 4 independent experiments; *p < 0.05. E, Western blot analysis of whole-cell homogenates and cytoskeletal extracts. The blots were developed with anti-paxillin antibody (pax). Note the increase in paxillin (pax) in the cytoskeletal fraction (Cytosk) of Aβ-treated neurons (Aβ). Total paxillin levels in whole-cell homogenates did not change. Similar tubulin levels (tub) confirmed equal loading. Con, Control. F, Densitometric quantification of paxillin in the cytoskeleton revealed a 680 ± 200% increase in Aβ-treated neurons grown on poly-l-lysine and a 210 ± 30% increase in control (Con) neurons grown on laminin (Lam).n = 9 independent experiments; *p < 0.05 relative to control by Student'st test.