Departments of 1Physiology and 2Microbiology and Immunology, The Brody School of Medicine, East Carolina University, Greenville, North Carolina 27858
Submitted 4 December 2003 ; accepted in final form 22 March 2004
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ABSTRACT |
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sciatic nerve; estrogen receptor; extracellular signal-regulated kinase
In the present study, we examined whether estrogen may influence retrograde axonal transport via ERK and/or PI3K signaling cascades in motoneurons. The effect of 17-estradiol was evaluated on the model of retrograde labeling of lumbar motoneurons in ovariectomized female mice after sciatic nerve axotomy. A mix of retrograde tracer (Fluorogold) and 17
-estradiol, in combination with an antagonist for ER ICI 182,780, an inhibitor of ERK1/2 pathway (U0126), an inhibitor of PI3K (LY-294002), or a protein synthesis inhibitor (cycloheximide), was applied to the proximal stump of the transected sciatic nerve for 24 h. Our results showed that 17
-estradiol applied directly onto the cut sciatic nerve produced a significant increase in the number of Fluorogold-labeled lumbar motoneurons. Estrogenic stimulation of retrograde labeling was inhibited by application of ICI 182,780, U0126, LY-294002, as well as cycloheximide. The present results indicate a role for estrogen, ERK, and PI3K signaling cascades in the uptake and retrograde transport of Fluorogold in motoneurons.
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MATERIALS AND METHODS |
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Two weeks after ovariectomy, the right sciatic nerve was cut in the midthigh, and a 7-mm Silastic tube with a 1.47-mm inner diameter was applied to the proximal nerve stump (28, 48). Afterward, the fluorescent dye Fluorogold or a mixture of Fluorogold with pharmacological agents was administered into the tube (7 µl total volume). The lower end of the tube was sealed with a petroleum jelly (Vaseline), the tube was glued to the surrounding skeletal muscles with tissue adhesive (3M Vetbond), and the incision was closed with wound clips. After application of Fluorogold for 24 h, animals were killed. In every experiment, we verified that the tube was still in place and that Fluorogold did not stain the adjacent tissue. Only the mice, which had the yellowish proximal nerve stump sited inside the tube (90% of animals), were employed in the study.
For immunohistochemical study, 2 wk after ovariectomy, the right sciatic nerves were crushed in the midthigh for 15 s with a fine hemostat (n = 3). In sham operations, right sciatic nerves were shortly exposed, the wounds were closed, and the mice were allowed to recover (17). The duration of the crush injury was 24 h. Animal protocols were approved by the East Carolina University Animal Care and Use Committee.
Retrograde labeling.
For retrograde labeling of motoneurons, 5% Fluorogold (Fluorochrom, Denver, CO) in vehicle was applied to the proximal stump of the nerve for 24 h. Retrograde labeling of motoneurons was performed to evaluate the efficiency of axonal transport in the presence of 10 nM 17-estradiol (Sigma) (40); 1 µM ICI 182,780 (40), an ER antagonist (Tocris); 100 µM U0126 (9), an inhibitor of mitogen-activated protein kinase kinase (MEK)1/2 (Cell Signaling); 100 µM LY-294002 (38), an inhibitor of PI3K (Calbiochem); and 10 µg/ml cycloheximide (29), an inhibitor of protein synthesis (Sigma). 17
-Estradiol, ICI 182,780, and U0126 were initially dissolved in 100% DMSO and further diluted with saline; thus the final concentration of DMSO was the same in all mixtures and did not exceed 2%, which has no effect on uptake and retrograde transport of Fluorogold (48). In all experiments, inhibitors were applied to the nerve stump 15 min before application of a mixture containing Fluorogold with an inhibitor, or Fluorogold with an inhibitor and 17
-estradiol. To evaluate whether systemic estrogen treatment might affect retrograde axonal transport, some mice were injected subcutaneously with 17
-estradiol (0.1 ml; 500 µg/kg) (25).
Morphometry of retrograde-labeled motoneurons. Twenty-four hours postsurgery, mice were euthanized and perfused with cold PBS, followed by cold 4% paraformaldehyde in PBS (pH 7.4). Lumbar spinal cords were removed and postfixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and embedded in Tris-buffered saline tissue-freezing medium (Triangle Biomedical Science, Durham, NC). Cryostat serial coronal 30-µm sections were analyzed with fluorescence microscopy by using a wideband ultraviolet filter. Fluorogold-labeled motoneurons in the ipsilateral side were recognized by their size, shape, and location (Fig. 1) and counted in every section without allowing for split nucleoli, according to the protocol described elsewhere (28, 48). The images were analyzed for the mean cross-sectional area of motoneurons by using ImageJ (NIH Image). We observed no difference in the mean cross-sectional area of labeled cells between control and treated animals, which is why we did not correct the neuronal counts using Abercrombie's formula. All values are represented as the means ± SE. Statistical analysis was performed with one-way ANOVA Newman-Keuls multiple-comparison test by using Prism (GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego, CA).
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Immunohistochemistry. All tissues for immunohistochemistry were collected 24 h after nerve crush. The staining was performed according to our laboratory protocol, as described previously (18). Mice were perfused with cold PBS, followed by cold 4% paraformaldehyde in PBS (pH 7.4). Sham sciatic nerves (at the midthigh level) and crushed nerves (a 5-mm piece with crush site in the middle) were removed and postfixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and embedded in Tris-buffered saline tissue-freezing medium (Triangle Biomedical Science). Frozen 10-µm longitudinal sections of sciatic nerves were processed for immunostaining. Rabbit phospho-ERK antibodies (Cell Signaling) were applied at 1:200 dilution. Sections were stained by using the Elite ABC kit (Vector Laboratories), and tissue antigens were visualized with a DAB substrate kit for peroxidase (Vector Laboratories). Densitometric analysis of the immunoprecipitate intensity was performed by using Kodak Digital Science 1D Image Analysis software (Eastman Kodak, Rochester, NY). The average value obtained from sham sciatic nerves was taken as 100%.
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RESULTS |
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Counts of radioactivity in both lumbar spinal cords and sciatic nerves were at background levels. The distal portion of the proximal stump (immersed in [3H]estradiol) had slightly higher scintillation counts compared with the proximal part. The counts on the right (injured) and left (contralateral) sides of the lumbar spinal cord were equal to the proximal portion of the sciatic nerve and did not differ between each other (Fig. 2).
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Effects of 17-estradiol, MEK1/2 inhibitor U0126, and PI3K inhibitor LY-294002 on retrograde labeling of motoneurons.
We examined systemic and local action of estrogen on retrograde labeling of lumbar motoneurons. To study the systemic effect of estrogen, mice (n = 4) were given a subcutaneous injection of 17
-estradiol following application of Fluorogold to the proximal nerve stump. The local effect of estrogen on retrograde labeling was established when a mixture of Fluorogold with 17
-estradiol was applied to the transected sciatic nerve (n = 4). Both systemic and local delivery of estrogen enhanced retrograde labeling of Fluorogold (Fig. 3A). After 24 h, the total number of Fluorogold-labeled motoneurons was nonsignificantly elevated in systemically treated mice (1,314 ± 155 vs. 963 ± 118; P < 0.1) and was significantly higher in mice with local application of estrogen (1,449 ± 159; P < 0.05) compared with control (963 ± 118), which had Fluorogold alone (n = 7).
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Local application of PI3K inhibitor LY-294002 (n = 3) to the proximal stump of the sciatic nerve had no direct effect on retrograde labeling. Indeed, the number of labeled motoneurons did not differ from control values (949 ± 130 vs. 963 ± 118) (Fig. 3B). At the same time, 17-estradiol mixed with LY-294002 (n = 3) did not increase the number of Fluorogold-labeled cells (891 ± 253) compared with control (963 ± 118; P > 0.05) or LY-294002-treated mice (949 ± 130; P > 0.05). Thus these results indicate that PI3K inhibitor LY-294002 blocks the estrogen-induced increase in retrograde labeling.
In contrast, MEK1/2 inhibitor U0126 (n = 4) markedly affected retrograde labeling. Application of U0126 alone caused a significant decrease in the number of labeled motoneurons (405 ± 57 vs. 963 ± 118, P < 0.05) (Fig. 3B). Estrogen coapplied to the nerve stump with U0126, 15 min after preincubation with the inhibitor (n = 4), failed to increase the retrograde labeling (353 ± 115 vs. 405 ± 57; P > 0.05).
The two selected inhibitors have shown a noticeably different impact on retrograde labeling. Obviously, the PI3K inhibitor did not have a direct effect on labeling compared with control, whereas the MEK1/2 inhibitor alone significantly reduced the number of Fluorogold-labeled cells. Coapplication of estrogen, in combination with any of the inhibitors, did not increase retrograde labeling. These data indicate a role of both MEK/ERK and PI3K signaling in regulation of peripheral nerve function.
Within the axon, the local translation of mRNA is an important source of proteins during axon growth (12). In this study, we investigated Fluorogold backlabeling, under condition of inhibited protein synthesis. Application of the protein synthesis inhibitor cycloheximide alone to the proximal stump of the nerve (n = 4) showed a significant decrease in the number of labeled motoneurons, compared with control (490 ± 93 vs. 963 ± 118; P < 0.05) (Fig. 3C). Estrogen applied to the proximal nerve stump after 15-min preincubation with cycloheximide (n = 4) partially removed the blocking effect of the protein synthesis inhibitor on retrograde labeling. The number of labeled motoneurons was not statistically different from that of control mice and constituted 594 ± 179.
The data indicate that the local inhibition of protein synthesis reduced retrograde transport of Fluorogold, whereas estrogen coapplied with cycloheximide partially negated the action of the cycloheximide.
Accumulation of phospho-ERK1/2 in the sciatic nerve 24 h after crush injury. Our previous study (18) has demonstrated a 1.8-fold increase in the level of activated ERK1/2 after crush injury of the sciatic nerve. The data obtained in this study showed that the MEK1/2 inhibitor significantly reduced retrograde labeling of motoneurons. To evaluate the possible involvement of ERK in axonal transport, we analyzed localization of phospho-ERK in the regenerating sciatic nerve 24 h after crush injury.
Staining of sham sciatic nerves revealed slightly immunopositive nerve fibers (Fig. 4A). In the injured nerve, the proximal part (above the crush site) had a weak staining of the nerve fibers as well. Interestingly, in the distal portion (below the crush site) of the nerve, we observed strongly ERK-positive regenerating axons (Fig. 4B).
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DISCUSSION |
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Retrograde axonal transport of target-derived neurotrophic factors is pivotal for survival, axon growth, and navigation during development and regeneration of motoneurons. Neurotrophins, such as nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3, are imported into the cell by receptor-mediated internalization and can affect cell function locally or be delivered to the perikarya by retrograde axonal transport (10, 13, 22, 24, 49).
Our laboratory's recent studies suggest a positive role of estrogen on regeneration of peripheral nerves (17, 18). Systemic delivery of the estrogen significantly enhanced regeneration of the sciatic nerve in ovariectomized female mice. Macroarray analysis in the lumbar spinal cord revealed upregulation of a wide array of genes promoting axonal growth, including genes encoding motor and cytoskeletal proteins, as well as kinesin motor protein C2, neuronal kinesin heavy chain, Rac1, and cortactin (18). Furthermore, we observed increased expression of genes for ER- and ER-
in axotomized lumbar motoneurons and accumulation of ERs in the regenerating axons.
Both genomic and nongenomic mechanisms of estrogen action could be implicated in the regulation of retrograde axonal transport. Our experiments with ER antagonist ICI 182,780 applied to the cut sciatic nerve demonstrated that the local inhibition of ERs blocked the effect of systemically delivered estrogen on retrograde delivery of Fluorogold. Therefore, estrogen potentiation of retrograde labeling was mediated via a local signaling mechanism. Indeed, the tracing of [3H]estradiol applied to proximal stump of the severed sciatic nerve showed no retrograde transport of [3H]estradiol to the soma of the lumbar motoneurons.
Interestingly, estrogen coapplied to the nerve stump with ICI 182,780 abolished the blocking effects of the ER antagonist on axonal transport. This may be a result of competitive interaction between estrogen and ICI 182,780, which confirms that estrogen action on retrograde labeling of motoneurons is mediated via local ERs.
Estrogen has been shown to activate PI3K (19) and ERK signaling cascades in neuronal cells (40). The PI3K activity plays an important role in neuronal polarity (38), retrograde axonal transport (48), and neuroprotection (14, 16). In sympathetic neuron cultures, the PI3K pathway was shown to be critical for neurotrophin retrograde signaling and neuronal survival (22). The ERK signaling cascade was shown to be involved in neuronal differentiation (2), local protein synthesis (29), dendrite formation (47), and cellular viability (4, 15). In addition, it has been demonstrated that PI3K and ERK pathways might cooperatively affect neuronal function (5) and regulate retrograde neurotrophin signaling via an endosome-based mechanism (10, 13, 49).
It has been demonstrated that estrogen cross talks with both cascades (19, 40). The estrogen-induced activation of the ERK-signaling pathway in neurons is well established, although the exact role of estrogen in ERK activation is far from clear. Recent evidence of ERK-dependent translation of mRNA within axons (29), and the fact that ERK can be transported in the anterograde (34) and retrograde (10) directions, suggest that ERK may regulate axonal transport in two aspects. It may activate the local synthesis of specific proteins and phosphorylate cytoskeletal and motor proteins. Thus both ERK and PI3K are likely to contribute to the internalization of Trk-containing endosomes and the maturation and retrograde transport of signaling vesicles critical for neuronal survival (13).
To investigate the possible mechanism of estrogen action on retrograde labeling, PI3K inhibitor (LY-294002) or MEK1/2 inhibitor (U0126) was applied to the proximal stump of the severed sciatic nerve alone or in the presence of estrogen. Interestingly, that PI3K inhibitor (LY-294002) did not impair the retrograde labeling in our experiments, whereas it attenuated retrograde signaling in sympathetic neuron culture (22). The discrepancy could be explained by using different experimental models. It is likely that the effect of LY-294002 in sympathetic neuron culture was somewhat stronger than on axotomized sciatic nerve in live animal. Another possible explanation is that the effect of LY-294002 on retrograde signaling could be more pronounced in sympathetic neurons, which depend on nerve growth factor for their survival, than in motoneurons. The failure of estrogen to increase retrograde labeling in the presence of U0126 and LY-294002 indicates the possible cross talk among estrogen, ERK, and PI3K signaling pathways in peripheral nerve fibers. Therefore, our observations suggest that estrogen may trigger synergistic effects of PI3K and MEK-ERK cascades on retrograde transport.
The recent data on local translation of mRNA within axons indicate that local protein synthesis is an essential event during nerve regeneration (12, 42, 43, 51). Based on this and the data that the estrogen-activated MEK-ERK cascade may affect local protein synthesis (29), we investigated the effects of the protein synthesis inhibitor cycloheximide on axonal transport. Cycloheximide alone significantly reduced the number of labeled motoneurons. Coapplication of estrogen with cycloheximide partially removed the blocking effect of the inhibitor on retrograde labeling of motoneurons. The data suggest that impaired local protein synthesis severely affects retrograde transport and that estrogen may support protein synthesis and/or stimulate axonal transport via activation of ERK. Thus the ER antagonist (ICI 182,780), inhibitors for MEK1/2 (U0126) and PI3K (LY-294002), and a protein synthesis inhibitor (cycloheximide) blocked the retrograde labeling at different levels (Fig. 6).
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Our observations demonstrated a role for estrogen, ERK, PI3K, and local protein synthesis in the retrograde labeling of axotomized lumbar motoneurons. Therefore, we propose that estrogen-induced potentiation of retrograde labeling of lumbar motoneurons is mediated via ERK and PI3K signaling pathways and is reliant on local protein synthesis.
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ACKNOWLEDGMENTS |
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This work was supported in part by Navy Grant N00014-01-1-0099 (to A.K. Murashov).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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