SCG10 is a JNK target in the axonal degeneration pathway

Abstract

Axons actively self-destruct following genetic, mechanical, metabolic, and toxic insults, but the mechanism of axonal degeneration is poorly understood. The JNK pathway promotes axonal degeneration shortly after axonal injury, hours before irreversible axon fragmentation ensues. Inhibition of JNK activity during this period delays axonal degeneration, but critical JNK substrates that facilitate axon degeneration are unknown. Here we show that superior cervical ganglion 10 (SCG10), an axonal JNK substrate, is lost rapidly from mouse dorsal root ganglion axons following axotomy. SCG10 loss precedes axon fragmentation and occurs selectively in the axon segments distal to transection that are destined to degenerate. Rapid SCG10 loss after injury requires JNK activity. The JNK phosphorylation sites on SCG10 are required for its rapid degradation, suggesting that direct JNK phosphorylation targets SCG10 for degradation. We present a mechanism for the selective loss of SCG10 distal to the injury site. In healthy axons, SCG10 undergoes rapid JNK-dependent degradation and is replenished by fast axonal transport. Injury blocks axonal transport and the delivery of SCG10, leading to the selective loss of the labile SCG10 distal to the injury site. SCG10 loss is functionally important: Knocking down SCG10 accelerates axon fragmentation, whereas experimentally maintaining SCG10 after injury promotes mitochondrial movement and delays axonal degeneration. Taken together, these data support the model that SCG10 is an axonal-maintenance factor whose loss is permissive for execution of the injury-induced axonal degeneration program.

Fig. 1.

SCG10 loss is an early marker of axonal injury. (A) Immunoblot analysis of endogenous SCG10 in cultured DRG axons with or without axotomy. The SCG10 level is decreased dramatically in the distal axons collected 3 h after axotomy compared with the level in the uncut control axons. Immunoblot against neuron-specific β3 tubulin confirms comparable amounts of protein loaded. (B) Axonal SCG10 is examined by immunostaining 3 h after axotomy in the DRG cultures infected with control or cytNmnat1-expressing lentivirus. The SCG10 level is decreased by axotomy to a similar extent in the distal axons of cytNmnat1-expressing neurons and in the control culture. β3 tubulin (green) labels microtubules in axons. (Scale bar, 50 µm.) (C) Three hours after sciatic nerve transection in adult mice, SCG10 levels were assayed by immunoblot in a control nerve and in the nerve segment distal to the axotomy. The SCG10 level is decreased significantly by axotomy. β3 tubulin is shown as a loading control. (D) SCG10 is lost selectively in the distal axons as shown by immunolabeling against SCG10 and β3 tubulin in the DRG cultures 3 h after axotomy. Arrows indicate the site of axotomy. SCG10 protein is accumulated in the proximal segment. (Scale bar, 50 µm.)

Fig. 2.

The SCG10 level is regulated by JNK in healthy and injured axons. (A) Immunostaining for SCG10 is shown in the cultured DRG axons labeled with anti-β3 tubulin antibody. Neurons were axotomized for 3 h, and either vehicle (DMSO) or the JNK inhibitor SP600125 (JNK i; 15 µM) was applied at the time of axotomy. SCG10 loss after axotomy is significantly reduced by inhibiting JNK activity. JNK inhibition also causes a dramatic increase in SCG10 level in noninjured axons. (Scale bar, 30 µm.) (B) Results from A were quantified for the axonal SCG10 levels normalized to the baseline SCG10 level (dotted line, no axotomy and vehicle). Axonal SCG10 levels were obtained by calculating the average SCG10 immunofluorescence within the β3 tubulin-positive area. n = 5, axotomy and vehicle; n = 5, axotomy and JNKi; n = 9, no axotomy and JNKi. *P < 0.01 for axotomy and JNK i vs. axotomy and vehicle (two-sample t test); **P < 0.005 for no axotomy and JNK i vs. baseline level (one-sample t test). Error bars represent SEM. (C) Axonal SCG10 degradation was examined by immunoblot analyses on the axonal lysates. (Top) SCG10 degradation was examined by inhibiting protein synthesis in DRG neurons by CHX treatment for the indicated time. (Middle) SCG10 turnover is slowed by inhibition of JNK with 15 µM SP600125 (JNK i). (Bottom) SCG10 turnover after axotomy was assessed at the indicated times after axotomy. β3 tubulin is shown as a loading control. (D) Quantification of the results shown in C. SCG10 levels are plotted as the fraction of the SCG10 level at time 0. The rate of SCG10 turnover is comparable in CHX-treated axons and injured distal axons, although the beginning of SCG10 loss appears delayed in CHX-treated axons. JNK inhibition results in a significant decrease in the SCG10 turnover rate. *P < 0.05, **P = 0.005 vs. CHX by t test. n = 3 for each condition. Error bars represent SEM.

Fig. 3.

JNK phosphorylation targets SCG10 for proteasomal degradation. (A) DRG cultures were axotomized (ax) and treated with DMSO (veh), 15 µM SP600125 (JNK i), or 20 µM MG132 for 3 h, and the distal axons were subjected to Western blot analysis to assess the gel mobility of SCG10. SP600125-treated axons preferentially preserve lower-molecular-weight SCG10, whereas MG132-treated axons preferentially preserve the higher-molecular-weight form. β3 tubulin is shown as a loading control. (B) In vitro treatment with phosphatase shows that the higher-molecular-weight SCG10 species accumulated by MG132 treatment is phosphorylated SCG10. Axonal extracts were obtained from the cultures that were axotomized, treated with MG132 for 3 h, and subsequently incubated with calf intestinal phosphatase. The amounts of GAPDH show equal loading. (C) The axonal levels of lentivirally expressed ven-SCG10 WT and ven-SCG10-AA were assayed after axotomy. Immunoblot with anti-SCG10 antibody shows both endogenous and lentivirally expressed ven-SCG10 (Upper). Ven-SCG10 WT is lost significantly in the distal axons by 6 h after axotomy, whereas ven-SCG10-AA is largely preserved. β3 tubulin is shown as a loading control (Lower). (D) JNK inhibition by 15 µM SP600125 (JNK i) attenuates the degradation of both wild-type SCG10 (ven-SCG10 WT) and alanine-mutant SCG10 (ven-SCG10-AA) after axotomy (Upper). β3 tubulin is shown as a loading control (Lower).

Fig. 4.

Anterograde axonal transport of SCG10. (A) Live imaging of Venus-tagged SCG10 in cultured DRG axons. (Upper) Four single frames with the same time interval are shown as representative images. Arrowhead indicates tracking of ven-SCG10 puncta. (Lower) The axon is visualized by maximum projection of all single frames in the time-lapse images. (Scale bar, 10 µm.) (B) A kymograph generated from the axon shown in A demonstrates that ven-SCG10 is transported predominantly in the anterograde direction. The kymograph corresponds to a 79-s live imaging. d, distance; t, time.

Fig. 5.

Knockdown of SCG10 accelerates axon degeneration after injury. (A) SCG10 was depleted by lentiviral expression of three different shRNA constructs (shRNA nos. 1, 2, and 3). SCG10 expression was restored by coexpressing rat SCG10 cDNA with the mouse-specific shRNA no. 1. Phase-contrast images show that axonal fragmentation is robust at 6 h after axotomy when SCG10 is knocked down; however, this effect is diminished when SCG10 expression is rescued. (Scale bar, 500 µm.) (B) Immunoblot for SCG10 shows effective knockdown of SCG10 by the lentiviral shRNAs and the rescue of the protein expression. β3 tubulin is shown as a loading control. (C) Quantification of the results in A. The degeneration index is a measure for fragmented axons. By ANOVA at 6 h, P < 0.001 for control vs. shRNA no. 1, 2, or 3; P < 0.01 for shRNA no. 1 vs. shRNA no. 1 + rescue. At 9 h, P < 0.05 for control vs. shRNA no. 1 or 2; P < 0.01 for, shRNA no. 1 vs. shRNA no. 1 + rescue. n = 9–15. Error bars represent SEM.

Fig. 6.

Shielding SCG10 from JNK phosphorylation delays axonal degeneration. (A) Axonal SCG10 levels were assayed with the lentiviral expression of control vector, nontagged SCG10 WT, or nontagged SCG10-AA following axotomy. Immunoblot for SCG10 shows that the level of lentivirally expressed SCG10 WT is decreased greatly by 6 h after axotomy, whereas SCG10-AA is largely preserved. β3 tubulin is shown as a loading control. (B) Quantification of the results in A. The levels of SCG10 at 6 h after axotomy are quantified as the fraction of the baseline SCG10 level (0 h after cut) in the respective lentiviral infection. Control, n = 4; SCG10 WT and SCG10-AA, n = 3; *P < 0.05, **P < 0.005 by ANOVA. Error bars represent SEM. (C) Results from A were quantified as relative levels of SCG10 normalized to that in the control at 0 h after cut. Numbers at the bottom of the bars represent time (in hours) after axotomy. Lentiviral expression of SCG10-AA leads to the preservation of SCG10 comparable to the level of endogenous SCG10 in unaxotomized axons up to 6 h after axotomy. Control, n = 4; SCG10 WT and SCG10-AA, n = 3; **P < 0.005, ***P < 0.001 by t test. Error bars represent SEM. (D) Phase-contrast images of axons were taken 12 h after axotomy from the DRG cultures infected with the indicated lentiviruses. Expression of SCG10-AA attenuates axon fragmentation. (Scale bar, 500 µm.) (E) Degeneration indices were obtained from the results in D and plotted against time after axotomy. At 0, 6, and 9 h after axotomy, n = 15–18; 12 h after axotomy, n = 10–12; 24 h after axotomy, n = 5–6; *P < 0.05, ***P < 0.001 vs. control lentivirus by ANOVA. Error bars represent SEM. n.s., not significant.

Fig. 7.

Axonal protection by expression of SCG10-AA is enhanced by JNK inhibition. (A) Axonal SCG10 levels were assayed with the lentiviral expression of control vector or nontagged SCG10-AA after axotomy with or without treatment of JNK inhibitor, 15 µM SP600125 (JNK i). Immunoblot with anti-SCG10 antibody shows that the levels of total SCG10 are maintained longer in the axons treated with JNK inhibitor. β3 tubulin is shown as a loading control. (B) Results in A are quantified as relative SCG10 levels normalized to the basal SCG10 level (control, vehicle-treated, 0 h after cut). n = 3; *P < 0.001 by ANOVA. Error bars represent SEM. (C) Axonal protection is extended by expressing SCG10-AA and inhibiting JNK activity. Axons are fragmented by 24 h after axotomy when treated with either lentiviral expression of SCG10-AA or JNK inhibitor. In contrast, simultaneously expressing SCG10-AA and inhibiting JNK leads to significant axonal preservation. (Scale bar, 500 µm.) (D) Quantification of the results in C; 0–24 h, n = 12; 56 h. n = 3 for control and vehicle; n = 8 for other conditions. Statistical significance is noted only for 24 h and 56 h after axotomy; by ANOVA, *P < 0.001 vs. control and vehicle; ⁺P < 0.001 vs. control and vehicle, vs. control and JNK inhibitor and vs. SCG10-AA and vehicle; ^#P < 0.05 vs. control and vehicle; ^#P < 0.01 vs. control and JNK inhibitor and vs. SCG10-AA and vehicle. Error bars represent SEM.

Fig. 8.

Mitochondrial transport is preserved by the expression of SCG10-AA. (A) Kymographs showing axonal transport of mitochondria labeled with mitochondrially targeted DsRed before axotomy (−Ax) or 1 h after axotomy (+Ax). The number of motile mitochondria decreases markedly after injury in control (vector-expressing) axons, whereas axons expressing SCG10-AA largely maintain mitochondrial transport. The kymographs correspond to 5 min of live imaging. Distal is to the right. d, distance; t, time. (B) Percent of motile mitochondria shown in A is quantified. n = 13–14; *P < 0.05, **P < 0.001 by ANOVA. Error bars represent SEM. n.s., not significant.

Fig. P1.

JNK-dependent SCG10 loss is involved in the axon degeneration pathway. (A) SCG10 is a labile protein whose degradation is regulated by JNK phosphorylation. Within 3 h after axotomy, wild-type SCG10 is lost rapidly in the distal axon segment. In contrast, SCG10 is transported anterogradely and accumulates in the proximal stump. By 9 h after injury, distal axons usually fragment. Treatment with JNK inhibitor (JNK i) attenuates SCG10 loss and delays axon degeneration. (B) Depleting SCG10 by knockdown accelerates axonal fragmentation after injury. (C) When two serines that are JNK phosphorylation sites are replaced by alanines (SCG10-S62A,S73A), SCG10 loss after injury is reduced significantly. The cross (X) indicates that JNK cannot phosphorylate the two mutated sites. Preserving SCG10 levels is sufficient to protect axons for up to 12 h after axotomy. SCG10 in the cell body has been omitted for clarity.