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First published online 25 August 2004
doi: 10.1242/jcs.01135

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Mitogen-activated protein kinase regulates neurofilament axonal transport

¹ Center Cell Neurobiology and Neurodegeneration Research, University of Massachusetts, Lowell, MA 01854, USA
² Department of Biological Sciences, University of Massachusetts, Lowell, MA 01854, USA
³ Department of Pharmacology, Vanderbilt University, Robinson Research Building, Nashville, TN 37232-8548, USA
⁴ Department of Pharmacology, College of Medicine, University of Tennessee Center for Health Sciences, 874 Union Avenue, Memphis, TN 38163, USA
⁵ Laboratory of Neurochemistry, NIH, NINDS, 36 Convent Drive, Bethesda, MD 20892, USA

View larger version (48K): [in a new window]   Fig. 1. Alteration of MAP kinase levels in NB2a/d1 cells. (A) Cultures were treated with either Provectin or ß-galactosidase or both together, followed by histochemical analysis for ß-galactosidase. Bright-field images of cells exposed for 4 hours to Provectin alone (Provec) or Provectin mixed with ß-galactosidase (Provec + ß-gal) are shown. The accompanying graph depicts the percentage of cells expressing ß-galactosidase activity in multiple fields of duplicate cultures; 100% of cells expressed ß-galactosidase activity following incubation with Provectin and ß-galactosidase, whereas 0% expressed activity following incubation with Provectin alone or with ß-galactosidase in the absence of Provectin. (B) Immunoblot analysis of homogenates of control cultures (Cont) and cultures treated for 4 hours with Provectin (Prov) and Provectin loaded with purified MAP kinase (Load), or for 24 hours with antisense oligonucleotides directed against MAP kinase (Anti) as described in Materials and Methods. Anti-MAP kinase recognizes two bands of 44 and 42 kDa. The accompanying graph presents densitometric analyses of immunoblots treated as indicated prior to harvest; note that treatment with Provectin and MAP kinase increased MAP kinase levels by approximately 35%, whereas treatment with antisense oligonucleotides reduced MAP kinase levels by nearly 40%.

View larger version (33K): [in a new window]   Fig. 2. MAP kinase alters intracellular NF phosphorylation. Immunoblot analyses of Triton-insoluble cytoskeletons from differentiated NB2a/d1 cells probed with SMI-31, RT97 and L3 (anti NF-L). Cells were immunoblotted following treatment with PD98059 (a MAP kinase activation inhibitor) (+, with PD98059; -, control); antisense (+) and sense (-) MAP kinase oligonucleotides; transfection with constructs expressing constitutively active and dominant-negative MAP kinase or following Provectin-mediated delivery of active or boiled MAP kinase or mock loaded as a control. Migratory positions of NF-H, NF-M and NF-L are indicated. The accompanying graphs present densitometric analyses, with values obtained from treated cultures expressed as % change compared to untreated cultures from the same experiment. Note that PD98059, antisense oligonucleotides and the dominant-negative MAP kinase decreased NF phospho-epitopes, whereas the constitutively active MAP kinase and intracellular delivery of active but not boiled MAP kinase increased NF phospho-epitopes. Note also that none of these treatments altered levels of NF-L immunoreactivity.

View larger version (45K): [in a new window]   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.

View larger version (55K): [in a new window]   Fig. 4. MAP kinase does not alter the distribution of mitochondria within axonal neuritis. Representative mitotracker-labelled images of NB2a/d1 cells treated for 2 hours with the MAP kinase inhibitor PD98059 or transfected 24 hours previously with constructs of constitutively active (Const-Act) or dominant-negative (Dom-Neg) MAP kinase are shown along with untreated controls. The accompanying graph presents densitometric analyses of the fluorescent intensity of mitotracker within the distal (Dist) compared to the proximal (Prox) half of axonal neurites, expressed as the percentage (mean±s.d.) within the distal neurite. Note that the distribution of mitotracker-labeled structures was not altered by the above treatments.

View larger version (73K): [in a new window]   Fig. 5. Manipulation of MAP kinase activity does not alter the transport or distribution of tau within axonal neuritis. (A) Representative fluorescent images of NB2a/d1 cells transfected 24 hours previously with a construct expressing CFP-htau40 with (+) and without (-) treatment with the MAP kinase inhibitor PD98059 for 2 hours. (B) Graph of the relative density of CFP-htau40 fluorescence, along with PHF-1, Tau-1 and 5E2 immunoreactivity within axonal neurites determined from 50-100 cells in duplicate cultures with or without PD98059 treatment for 2 hours. Note that PD98059 did not alter the distribution of newly transported (CFP-tagged) tau within axonal neurites, nor that of phospho-dependent (PHF-1, Tau-1) or independent (5E2) tau epitopes.

View larger version (74K): [in a new window]   Fig. 6. Manipulation of MAP kinase activity does not alter neurite length or alter steady state levels of actin and tubulin, but does alter C-terminal NF phosphorylation within axonal neuritis. (A) Fluorescent images of axonal neurites of NB2a/d1 cells following labeling with rhodamine-conjugated phalloidin to label actin (Actin) or an anti-tubulin antibody (Tubulin). The accompanying graph shows the relative density of actin and tubulin fluorescence along axonal neurites ±2 hours' treatment with PD98059 determined for 25-50 cells (mean±s.d.); note that there is no significant difference in the presence or absence of PD98059. (B) Cells double-labeled with an antibody against NF-L and the phospho-dependent anti-NF antibody RT97. The graph shows the ratio of RT97 density/NF-L density (as indices of phospho-NFs and total NFs, respectively) before and after a 2-hour treatment with PD98059 (mean±s.d.). Treatment with PD98059 induced a specific reduction in phospho-NFs. (C) The length (mean±s.d.) of 25-50 axonal neurites in duplicate cultures following treatment with PD98059 or transfection with constitutively active (Const-Act) or dominant-negative (Dom-neg) MAP kinase. Axonal neurite length was not altered by manipulation of MAP kinase activity.

View larger version (84K): [in a new window]   Fig. 7. Inhibition of MAP kinase activity inhibits NF axonal transport. (A,C,D) Sequentially captured images of axonal neurites of GFP-transfected NB2a/d1 cells (A,C) or mitotracker-stained cells (D) captured at the indicated intervals (in minutes) before and after addition of the MAP kinase inhibitor PD98059 (time of addition indicated by arrow). For each axonal neurite, one of the panels captured before PD98059 addition was false-colored green and a subsequent image was false-colored red and these images were then merged (indicated by B for before); the same procedure was carried out for two images captured after addition of PD98059 (indicated by A for after). Superimposing structures appear in yellow/orange, whereas non-superimposing structures remain red or green. This provides an additional index of the relative amount of moving versus stationary structures. Note that the majority of GFP-tagged structures do not superimpose prior to addition of PD98059, but do so following its addition (A), that PD98059 did not induce a similar increase in superimposing mitotracker-labeled structures (C) and that DMSO (carrier for PD98059) did not increase the number of superimposing GFP-tagged structures. Comparisons of images in overall sequences also indicate that PD98059 stopped the movement of GFP-tagged filamentous and particulate structures (A), did not affect the movement of mitotracker-labeled structures (C), and that DMSO did not affect the movement of GFP-tagged structures (D). (B) Percentage of filamentous or particulate GFP-tagged structures exhibiting net anterograde, retrograde or lack of net movement prior to (con, control) and following a 15-minute treatment with PD98059 (PD), olomoucine (Olo) or SB202190 (SB) as indicated. Note that PD98059 and olomoucine but not SB202190 dramatically increased the percentage of non-moving GFP-tagged structures. (E, F) Percentage of GFP-tagged (E) or mitotracker-labeled (F) particles exhibiting net motion in either the anterograde or retrograde direction (mov, `moving') or no net motion (non mov, `non moving') compiled from 5-10 individual axons. Note that the carrier DMSO (Con) and PD98059 itself did not alter the translocation of mitotracker-labeled particles, whereas olomoucine stopped translocation of approximately 80% of mitotracker-labeled particles.

View larger version (61K): [in a new window]   Fig. 8. SB202190 and olomoucine exert differential effects on NF transport in NB2a/d1 cells. (A-C) Sequential images of axonal neurites of GFP-M-transfected cells captured at the indicated intervals (in minutes) before and after addition of SB202190 (A) or olomoucine (B,C). The time of addition is indicated by the arrow in each case. (C) Images of cells 5-15 minutes after addition of olomoucine and mitotracker. Axonal neurites were imaged under fluorescent optics to visualize GFP and rhodamine optics to visualize mitotracker (Mito). False-color and merged images were prepared as described in the legend for Fig. 7 and in Materials and Methods. Note that SB202190 did not impair the transport of GFP-tagged structures (A). Note also that olomoucine inhibited transport of GFP-tagged structures, but did not inhibit transport of mitotracker-labeled structures in the same neurites (B,C).

View larger version (78K): [in a new window]   Fig. 9. Long-term inhibition of MAP kinase activity induces net retrograde NF transport. (A-C,E,G) GFP-transfected (A-C,G) and mitotracker-labeled (E) NB2a/d1 cells sequentially captured at the indicated intervals following treatment with PD98059. Selected GFP-tagged structures undergoing retrograde transport are indicated by arrows; not all such structures are indicated. (D) Data derived from over 100 individual GFP-tagged structures from multiple cultures from three experiments. Note that this more prolonged treatment with PD98059 induces a shift from net anterograde to net retrograde transport of GFP-tagged structures. Note that treatment with PD98059 for 3 hours did not reduce the overall level of mitotracker-labeled structures within axonal neurites (E, F). GC in panel C denotes the growth cone (Hillock). Scale bar in C (refers to all panels), 10 µm.

View larger version (57K): [in a new window]   Fig. 10. Inhibition of MAP kinase affects the distribution of normal and constitutively phosphorylated NF-H within axonal neurites equally. (A) Immunoprecipitation with anti-NF polyclonal antibody R39 from cultures transfected 24 hours previously with GFP-Hwt (wt) or GFP-Hasp (Asp) and probed with anti-GFP antibody; note the presence of GFP-immunoreactive species migrating at 200 kDa. (B) Representative images of cells transfected with GFP-Hwt or GFP-Hasp as indicated, before and after 2 hours' treatment with PD98059; images before and after treatment are of different cells. (C) Densitometric analysis of the ratio of GFP fluorescence in the distal versus the central third of axonal neurites from 10-20 individual cells from two independent experiments before and after a 2-hour treatment with PD98059. The lower graph shows the percentage reduction in GFP signal in the distal third of axonal neurites for each construct following a 2-hour treatment with PD98059, obtained by dividing the ratio before to that after treatment. Consistent with prior reports (see text), the ratio of GFP signal in distal/central axons is lower in cells transfected with GFP-Hasp compared to GFP-Hwt; however, note that 2 hours' treatment with PD98059 reduces this ratio by the same percentage for both constructs. A similar impression is obtained by visual inspection of the cells in panel B. (D) Sequentially captured images of representative axonal neurites of a cell transfected with GFP-Hasp at 15-second intervals commencing 15 minutes after addition of PD98059. The merged image (M) was prepared from the 0 and 30 second images as described in the legend for Fig. 7. Note that many structures in the merged image do not superimpose indicating continued net translocation following the addition of PD98059. (E) The percentage of filamentous or particulate GFP-tagged structures exhibiting net anterograde, retrograde or lack of net movement prior to (dark-shaded bars) and following (pale-shaded bars) a 15-minute treatment with PD98059 from 10-20 individual axons from two independent experiments for each construct quantified as described in Materials and Methods and in the legend to Fig. 7. Images of cells transfected with GFP-Hwt and of cells transfected with GFP-Hasp prior to the addition of PD98059 are not presented because, as indicated by quantification of particles (E) they are essentially identical to those presented for GFP-M in Fig. 7.