Figure 3.

Conditioned medium induces NFATc4-aberrant nuclear translocation in wild-type cultured neurons. A, Schematic representation of AKAP79 inhibitory peptide for CaN (A1) and AAV2 viral vectors (AAV–CMV/CBA–WPRE) with AKAP79 inhibitory peptide (A2). B, Aβ depletion or CaN inhibition with AKAP79 inhibitory peptide prevents TgCM-induced NFATc4 activation as seen in cultures stained with NFATc4 (red), MAP2 (green), and Hoechst nuclear counterstain (blue) from each experimental condition. C, Quantification of NFATc4-aberrant nuclear translocation induced by TgCM in wild-type cultured neurons. The ratio of nucleus to cytoplasm is shown. TgCM causes an increase in the nucleus/cytoplasm ratio of NFATc4 that is dependent on the presence of Aβ (because anti-Aβ antibody 3D6 prevents the effect). Inhibition of CaN activation by AKAP79 peptide also prevented the increase in the nucleus/cytoplasm ratio of NFATc4 (n > 40 cells). Data represent mean ± SD. *p < 0.05. D, Quantification of NFATc4-aberrant nuclear translocation induced by different SEC fractions in wild-type cultured neurons. Application of SEC fractions 6–7 (sAPP fraction) from either TgCM or wtCM caused no significant difference on the nucleus/cytoplasm ratio of NFATc4. However, application of SEC fractions 18–19 (Aβ fraction) of TgCM onto wild-type neurons for 24 h caused significant increase in translocation of NFATc4 to the nucleus, but no changes were observed in neurons applied with the same SEC fractions of wtCM. Immunodepletion of Aβ from the fractions 18–19 of TgCM with 3D6 prevented the increase in the nucleus/cytoplasm ratio of NFATc4. n > 35 cells. Data represent mean ± SD; *p < 0.05. ITR, Inverted terminal repeat.