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Fig. 3. MAP kinase inhibits accumulation of NFs in axonal neuritis. (A) Representative images of the various forms in which GFP-M is detected 24 hours after transfection of NB2a/d1 cells that had received dbcAMP for 2 days prior to transfection. As shown previously (Yabe et al., 2001b), most axonal neurites under these conditions display a centrally situated NF bundle that is prominently labeled with GFP along its length (`Filamentous'), some axonal neurites displayed only diffuse fluorescence within neurites (`Diffuse'), or smaller filamentous and punctate structures in addition to the filamentous bundle (`Mixed'). (B) Representative fluorescence images of cells treated with sense- or antisense-oriented MAP kinase oligonucleotides as indicated. Note the association of GFP with the NF bundle along the entire axonal length in cells receiving sense oligonucleotides, yielding a filamentous image and its absence in the distal neurite region in the presence of antisense oligonucleotides, yielding a diffuse pattern. (C) Percentage (mean±s.e.m.) of cells in which GFP-M was associated with the NF bundle in the distal third of the axonal length (as described in Materials and Methods) following treatment under various conditions as indicated in Fig. 2 and with SB202190, a p38 MAP kinase inhibitor. Note that PD98059, but not its carrier (DMSO) alone, antisense but not sense oligonucleotides and dominant-negative MAP kinase had reduced levels compared to the control. Note also that PD98059 did not prevent the increase induced by constitutively active MAP kinase.
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