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Sunday Driver links axonal transport to damage signaling.

Cavalli V, Kujala P, Klumperman J, Goldstein LS - J. Cell Biol. (2005)

Bottom Line: We found that syd and JNK3 are present on vesicular structures in axons, are transported in both the anterograde and retrograde axonal transport pathways, and interact with kinesin-I and the dynactin complex.Finally, we found that injury induces an enhanced interaction between syd and dynactin.Thus, a mobile axonal JNK-syd complex may generate a transport-dependent axonal damage surveillance system.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Neurons transmit long-range biochemical signals between cell bodies and distant axonal sites or termini. To test the hypothesis that signaling molecules are hitchhikers on axonal vesicles, we focused on the c-Jun NH2-terminal kinase (JNK) scaffolding protein Sunday Driver (syd), which has been proposed to link the molecular motor protein kinesin-1 to axonal vesicles. We found that syd and JNK3 are present on vesicular structures in axons, are transported in both the anterograde and retrograde axonal transport pathways, and interact with kinesin-I and the dynactin complex. Nerve injury induces local activation of JNK, primarily within axons, and activated JNK and syd are then transported primarily retrogradely. In axons, syd and activated JNK colocalize with p150Glued, a subunit of the dynactin complex, and with dynein. Finally, we found that injury induces an enhanced interaction between syd and dynactin. Thus, a mobile axonal JNK-syd complex may generate a transport-dependent axonal damage surveillance system.

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JNK is activated locally by sciatic nerve injury. (A) Sciatic nerve ligation was performed as in Fig. 3. Mice were killed 3, 6, or 8 h after surgery. DRGs on the ligated and contralateral unligated sides were dissected and analyzed by SDS-PAGE. Levels of P-JNK (closed squares), JNK1 (open circles), and JNK3 (open triangles) were normalized to tubulin, and the ratios between ligated and unligated were measured. Results represent mean ± SEM (n = 4). (B) Sciatic nerves were ligated unilaterally at the midpoint and stained for P-JNK. White arrows show ligation site. (C) Whole ligated nerve (lig), sham operated nerve (sham), and contralateral unligated (ul) nerve were dissected and homogenized, and extracts were analyzed by SDS-PAGE and Western blot (Western blot is representative of seven independent experiments; only the 6-h time point is shown). (D) Quantification of the relative ratio P-JNK/JNK3 between ligated and unligated nerves is shown ± SEM (n = 7). The P-JNK/JNK3 ratio in unligated nerve was normalized to 1. The level of P-JNK increased threefold in the ligated nerves after 1, 3, and 6 h. (E) Sciatic nerve cross sections ∼200 μm proximal or distal of the ligation site were stained for P-JNK and S100. Arrows, P-JNK staining in axon; arrowheads, P-JNK staining in Schwann cells. (F) Ligated and unligated nerves were subjected to high speed centrifugation. Soluble (S) and membrane-bound fractions (P) were analyzed by Western blot. The ratio P-JNK/JNK3 was determined as in B and shown as mean ± SEM (n = 4). Bars: (B) 100 μm; (E) 20 μm.
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fig5: JNK is activated locally by sciatic nerve injury. (A) Sciatic nerve ligation was performed as in Fig. 3. Mice were killed 3, 6, or 8 h after surgery. DRGs on the ligated and contralateral unligated sides were dissected and analyzed by SDS-PAGE. Levels of P-JNK (closed squares), JNK1 (open circles), and JNK3 (open triangles) were normalized to tubulin, and the ratios between ligated and unligated were measured. Results represent mean ± SEM (n = 4). (B) Sciatic nerves were ligated unilaterally at the midpoint and stained for P-JNK. White arrows show ligation site. (C) Whole ligated nerve (lig), sham operated nerve (sham), and contralateral unligated (ul) nerve were dissected and homogenized, and extracts were analyzed by SDS-PAGE and Western blot (Western blot is representative of seven independent experiments; only the 6-h time point is shown). (D) Quantification of the relative ratio P-JNK/JNK3 between ligated and unligated nerves is shown ± SEM (n = 7). The P-JNK/JNK3 ratio in unligated nerve was normalized to 1. The level of P-JNK increased threefold in the ligated nerves after 1, 3, and 6 h. (E) Sciatic nerve cross sections ∼200 μm proximal or distal of the ligation site were stained for P-JNK and S100. Arrows, P-JNK staining in axon; arrowheads, P-JNK staining in Schwann cells. (F) Ligated and unligated nerves were subjected to high speed centrifugation. Soluble (S) and membrane-bound fractions (P) were analyzed by Western blot. The ratio P-JNK/JNK3 was determined as in B and shown as mean ± SEM (n = 4). Bars: (B) 100 μm; (E) 20 μm.

Mentions: Previously, peripheral nerve transection in rat was shown to induce JNK activation in neuronal cell bodies (Kenney and Kocsis, 1998). To confirm that mouse sciatic nerve injury provoked by ligation also induces activation of JNK in neuronal cell bodies, we dissected and analyzed extracts of dorsal root ganglia (DRGs), which are enriched for sensory neuron cell bodies, 3, 6, and 8 h after sciatic nerve ligation. We observed an ∼1.6-fold increase in the level of P-JNK on the ligated side compared with the contralateral unligated side at the 6 and 8 h time points, whereas no increase was detected at the 3-h time point. The levels of JNK1 and JNK3 were unchanged (Fig. 5 A). Similarly to syd and JNK3, P-JNK accumulates on both proximal and distal sides of the ligature, whereas only very faint staining was observed in the unligated nerve (Fig. 5 B). To assess directly whether or not the amount of activated JNK also increased locally upon nerve injury, sciatic nerves were ligated for 6 h, and the amount of P-JNK and JNK3 detected in ligated versus unligated nerves were compared biochemically (Fig. 5 C). Although the amount of P-JNK is increased in the ligated nerve, the levels of JNK1 and JNK3 remain constant (tubulin is used as a loading control). Sham surgery controls indicate that the increase in P-JNK is due to axonal injury induced by ligation rather than the surgery procedure. We measured the ratio between P-JNK and JNK3 at 1, 3, and 6 h after ligation (normalized to 1 in the unligated nerve). We observed a threefold increase after 1 h of ligation (Fig. 5 D), with no further significant increase after 3 or 6 h. It is striking that the increase in P-JNK does not appear in the DRGs until the 6-h time point, whereas we observed an increase in P-JNK levels locally in the sciatic nerve after 1 h of ligation. This delay suggests that a retrogradely transported signal is mediating JNK activation in the cell bodies and might depend on local JNK activation. With an estimated retrograde transport velocity of ∼2 μm/s, a molecule traveling from the injury site would require ∼4 h to reach the DRGs, located ∼3 cm away. Our results are in agreement with previous observations (Kenney and Kocsis, 1998), where the delay observed between axotomy and JNK activation in the DRGs corresponds to a signal transmission velocity in the 2 to 5-μm/s range.


Sunday Driver links axonal transport to damage signaling.

Cavalli V, Kujala P, Klumperman J, Goldstein LS - J. Cell Biol. (2005)

JNK is activated locally by sciatic nerve injury. (A) Sciatic nerve ligation was performed as in Fig. 3. Mice were killed 3, 6, or 8 h after surgery. DRGs on the ligated and contralateral unligated sides were dissected and analyzed by SDS-PAGE. Levels of P-JNK (closed squares), JNK1 (open circles), and JNK3 (open triangles) were normalized to tubulin, and the ratios between ligated and unligated were measured. Results represent mean ± SEM (n = 4). (B) Sciatic nerves were ligated unilaterally at the midpoint and stained for P-JNK. White arrows show ligation site. (C) Whole ligated nerve (lig), sham operated nerve (sham), and contralateral unligated (ul) nerve were dissected and homogenized, and extracts were analyzed by SDS-PAGE and Western blot (Western blot is representative of seven independent experiments; only the 6-h time point is shown). (D) Quantification of the relative ratio P-JNK/JNK3 between ligated and unligated nerves is shown ± SEM (n = 7). The P-JNK/JNK3 ratio in unligated nerve was normalized to 1. The level of P-JNK increased threefold in the ligated nerves after 1, 3, and 6 h. (E) Sciatic nerve cross sections ∼200 μm proximal or distal of the ligation site were stained for P-JNK and S100. Arrows, P-JNK staining in axon; arrowheads, P-JNK staining in Schwann cells. (F) Ligated and unligated nerves were subjected to high speed centrifugation. Soluble (S) and membrane-bound fractions (P) were analyzed by Western blot. The ratio P-JNK/JNK3 was determined as in B and shown as mean ± SEM (n = 4). Bars: (B) 100 μm; (E) 20 μm.
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Related In: Results  -  Collection

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fig5: JNK is activated locally by sciatic nerve injury. (A) Sciatic nerve ligation was performed as in Fig. 3. Mice were killed 3, 6, or 8 h after surgery. DRGs on the ligated and contralateral unligated sides were dissected and analyzed by SDS-PAGE. Levels of P-JNK (closed squares), JNK1 (open circles), and JNK3 (open triangles) were normalized to tubulin, and the ratios between ligated and unligated were measured. Results represent mean ± SEM (n = 4). (B) Sciatic nerves were ligated unilaterally at the midpoint and stained for P-JNK. White arrows show ligation site. (C) Whole ligated nerve (lig), sham operated nerve (sham), and contralateral unligated (ul) nerve were dissected and homogenized, and extracts were analyzed by SDS-PAGE and Western blot (Western blot is representative of seven independent experiments; only the 6-h time point is shown). (D) Quantification of the relative ratio P-JNK/JNK3 between ligated and unligated nerves is shown ± SEM (n = 7). The P-JNK/JNK3 ratio in unligated nerve was normalized to 1. The level of P-JNK increased threefold in the ligated nerves after 1, 3, and 6 h. (E) Sciatic nerve cross sections ∼200 μm proximal or distal of the ligation site were stained for P-JNK and S100. Arrows, P-JNK staining in axon; arrowheads, P-JNK staining in Schwann cells. (F) Ligated and unligated nerves were subjected to high speed centrifugation. Soluble (S) and membrane-bound fractions (P) were analyzed by Western blot. The ratio P-JNK/JNK3 was determined as in B and shown as mean ± SEM (n = 4). Bars: (B) 100 μm; (E) 20 μm.
Mentions: Previously, peripheral nerve transection in rat was shown to induce JNK activation in neuronal cell bodies (Kenney and Kocsis, 1998). To confirm that mouse sciatic nerve injury provoked by ligation also induces activation of JNK in neuronal cell bodies, we dissected and analyzed extracts of dorsal root ganglia (DRGs), which are enriched for sensory neuron cell bodies, 3, 6, and 8 h after sciatic nerve ligation. We observed an ∼1.6-fold increase in the level of P-JNK on the ligated side compared with the contralateral unligated side at the 6 and 8 h time points, whereas no increase was detected at the 3-h time point. The levels of JNK1 and JNK3 were unchanged (Fig. 5 A). Similarly to syd and JNK3, P-JNK accumulates on both proximal and distal sides of the ligature, whereas only very faint staining was observed in the unligated nerve (Fig. 5 B). To assess directly whether or not the amount of activated JNK also increased locally upon nerve injury, sciatic nerves were ligated for 6 h, and the amount of P-JNK and JNK3 detected in ligated versus unligated nerves were compared biochemically (Fig. 5 C). Although the amount of P-JNK is increased in the ligated nerve, the levels of JNK1 and JNK3 remain constant (tubulin is used as a loading control). Sham surgery controls indicate that the increase in P-JNK is due to axonal injury induced by ligation rather than the surgery procedure. We measured the ratio between P-JNK and JNK3 at 1, 3, and 6 h after ligation (normalized to 1 in the unligated nerve). We observed a threefold increase after 1 h of ligation (Fig. 5 D), with no further significant increase after 3 or 6 h. It is striking that the increase in P-JNK does not appear in the DRGs until the 6-h time point, whereas we observed an increase in P-JNK levels locally in the sciatic nerve after 1 h of ligation. This delay suggests that a retrogradely transported signal is mediating JNK activation in the cell bodies and might depend on local JNK activation. With an estimated retrograde transport velocity of ∼2 μm/s, a molecule traveling from the injury site would require ∼4 h to reach the DRGs, located ∼3 cm away. Our results are in agreement with previous observations (Kenney and Kocsis, 1998), where the delay observed between axotomy and JNK activation in the DRGs corresponds to a signal transmission velocity in the 2 to 5-μm/s range.

Bottom Line: We found that syd and JNK3 are present on vesicular structures in axons, are transported in both the anterograde and retrograde axonal transport pathways, and interact with kinesin-I and the dynactin complex.Finally, we found that injury induces an enhanced interaction between syd and dynactin.Thus, a mobile axonal JNK-syd complex may generate a transport-dependent axonal damage surveillance system.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Neurons transmit long-range biochemical signals between cell bodies and distant axonal sites or termini. To test the hypothesis that signaling molecules are hitchhikers on axonal vesicles, we focused on the c-Jun NH2-terminal kinase (JNK) scaffolding protein Sunday Driver (syd), which has been proposed to link the molecular motor protein kinesin-1 to axonal vesicles. We found that syd and JNK3 are present on vesicular structures in axons, are transported in both the anterograde and retrograde axonal transport pathways, and interact with kinesin-I and the dynactin complex. Nerve injury induces local activation of JNK, primarily within axons, and activated JNK and syd are then transported primarily retrogradely. In axons, syd and activated JNK colocalize with p150Glued, a subunit of the dynactin complex, and with dynein. Finally, we found that injury induces an enhanced interaction between syd and dynactin. Thus, a mobile axonal JNK-syd complex may generate a transport-dependent axonal damage surveillance system.

Show MeSH
Related in: MedlinePlus