Microtubule-perturbing drugs inhibit neuronal transport of NF-κB.

(A) Effect of microtubule-perturbing drugs on the organization of neuronal microtubule network. Microtubule network was visualized by α−tubulin immunostaining. Treatment of hippocampal neurons with 200 nM colchicine or 100 nM vincristine resulted in efficient depolymerisation, as shown by the diffuse tubulin staining and the loss of the well-organized microtubule patterns seen in untreated cells.(B) Hippocampal neurons treated with 300 µM glutamate for 5 min, either alone or after pre-treatment with 200 nM colchicine or 100 nM vincristine for 30 min were fixed (90 min after glutamate exposure) and visualised by SYTOX nuclear staining (green) and anti-NF-κB p65 immunofluorescence (red) to monitor neuronal transport of NF-κB (C) Quantification of nuclear/dendritic ratio of α-p65 fluorescence in neurons with functional or disrupted microtubules. Note that microtubule-perturbing drugs impaired dendritic to nuclear redistribution of NF-κB p65 after glutamate stimulation. (D) Reporter gene assay showed reduced NF-κB-dependent transcription activity in neurons with not functional microtubules. (E) In order to confirm the data obtained by microscopy, the subcellular localization of p65 was examined by cell fractionation and Western blotting. 250 µg of soluble protein from cytoplasmic extracts were immunoblotted for p65 protein level. In agreement, with the above experiments, pre-treatment with vincristine or colchicines before exposure to glutamate resulted in reduced relocation of p65 from cytoplasm to the nucleus.