Distinct roles of c-Jun N-terminal kinase isoforms in neurite initiation and elongation during axonal regeneration

Abstract

c-Jun N-terminal kinases (JNKs) (comprising JNK1-3 isoforms) are members of the MAPK (mitogen-activated protein kinase) family, activated in response to various stimuli including growth factors and inflammatory cytokines. Their activation is facilitated by scaffold proteins, notably JNK-interacting protein-1 (JIP1). Originally considered to be mediators of neuronal degeneration in response to stress and injury, recent studies support a role of JNKs in early stages of neurite outgrowth, including adult axonal regeneration. However, the function of individual JNK isoforms, and their potential effector molecules, remained unknown. Here, we analyzed the role of JNK signaling during axonal regeneration from adult mouse dorsal root ganglion (DRG) neurons, combining pharmacological JNK inhibition and mice deficient for each JNK isoform and for JIP1. We demonstrate that neuritogenesis is delayed by lack of JNK2 and JNK3, but not JNK1. JNK signaling is further required for sustained neurite elongation, as pharmacological JNK inhibition resulted in massive neurite retraction. This function relies on JNK1 and JNK2. Neurite regeneration of jip1(-/-) DRG neurons is affected at both initiation and extension stages. Interestingly, activated JNKs (phospho-JNKs), as well as JIP1, are also present in the cytoplasm of sprouting or regenerating axons, suggesting a local action on cytoskeleton proteins. Indeed, we have shown that JNK1 and JNK2 regulate the phosphorylation state of microtubule-associated protein MAP1B, whose role in axonal regeneration was previously characterized. Moreover, lack of MAP1B prevents neurite retraction induced by JNK inhibition. Thus, signaling by individual JNKs is differentially implicated in the reorganization of the cytoskeleton, and neurite regeneration.

Figure 1.

JNK activation in central primary sensory fibers undergoing sprouting in response to a peripheral lesion. Shown are confocal microscope images. A, P-JNK immunostaining (red) in dorsal horn of adult mouse spinal cord, 1 week after unilateral sciatic nerve transection. P-JNK is detected in fibers in lamina II ipsilateral to the lesion (right), compared with the contralateral side (left), devoid of P-JNK staining. B–D, Higher magnification of the area marked by a white square in A reveals colocalization of P-JNK (red) with phosphorylated MAP1B (green) within the same fibers (arrowheads). The insets show single optical plane images. D, Dorsal; LII, lamina II; WM, white matter. Scale bars: A, 100 μm; B–D, insets, 40 μm.

Figure 2.

JNK activation is required for both neuritogenesis and sustained extension of regenerating neurites. A–D, P-JNK and tubulin immunolabeling of DRG neurons cultured for 48 h. P-JNK is found in soma (including the nucleus), axon shafts, and growth cones. E, Quantification of neurite-bearing neurons cultured for 24 and 48 h, without (gray bars) or with chronic SP600125 treatment (black bars), reveals that neurite formation is blocked by JNK inhibition (n ≥ 446). F, G, Neurons treated with or without SP600125 and visualized by tubulin immunostaining. JNK inhibition induces loss of growth cones and leads to retraction bulb formation and trailing remnants along neurites (arrowheads); insets show higher magnifications of a typical growth cone under control conditions and a drug treatment-induced retraction bulb. H, I, Quantitative analysis reveals that JNK inhibition decreases total neuritic and longest neurite lengths of regenerating neurons (n ≥ 55). J, K, Distribution diagrams showing the percentage of neurons exhibiting a given total neuritic length and length of the longest neurite. Whereas the majority of neurons cultured under control conditions displays a total neurite length >3500 μm and a longest neurite >400 μm, JNK inhibition induces a shift of both values toward neurons with shorter neurites. L, M, Frames of time-lapse recordings illustrating the typical response of neurites to JNK inhibition: Before SP600125 addition (t = 60 min), growth cones (arrowheads) are highly dynamic and neurites elongate steadily. Shortly after drug addition, growth cones collapse, and neurites stop extending and start to retract. Scale bars: A, C, F, G, L, M, 50 μm; B, D, 15 μm. ***p < 0.001. Error bars indicate SEM.

Figure 3.

Differential involvement of JNK isoforms in neurite initiation and elongation. A, Western blots of protein extracts from wild-type and JNK-deficient mice DRG neurons, cultured 48 h, reacted with antibodies specific for individual JNK proteins. In regenerating wild-type neurons, all isoforms are expressed. B, Quantification of neurons bearing neurites after 24 and 48 h of culture: At 24 h, jnk2^−/− and jnk3^−/− neurons exhibit a strong decrease in the percentage of neurons with neurites compared with wild-type neurons. At 48 h, only jnk2^−/− neurons still display a slight but significant difference. Neurite initiation is not affected in regenerating jnk1^−/− neurons (n ≥ 297). C, D, Quantification of total neurite length and length of the longest neurite for wild-type versus JNK-deficient neurons without (gray bars) or with SP600125 treatment (black bars). Total neurite lengths for jnk1^−/− and jnk2^−/−, but not jnk3^−/− neurons, are decreased compared with wild-type neurons; the average length of the longest neurite is significantly affected only by lack of JNK2. SP600125 treatment induces an additional reduction in total neurite length for neurons derived from all three jnk-ko mice, but induces a specific decrease in length of the longest neurite only in jnk3^−/− neurons (n ≥ 50). E, F, The effect on neurite length of JNK1 and JNK2, but not JNK3 deficiency, is reflected in a leftward shift in the distribution diagrams for total neurite length; values for the length of the longest neurite per neuron display a leftward shift only for jnk2^−/− neurons; those for jnk1^−/− and jnk3^−/− neurons are similar to wild type. ***p < 0.001; **p < 0.01; *p < 0.05. Error bars indicate SEM.

Figure 4.

Lack of JNK1 and JNK2 affects axonal elongation rate. A, Mean speed of growth cone advancement and retraction of wild-type and JNK-deficient DRG neurons. Note that jnk1^−/− and jnk2^−/−, but not jnk3^−/−, neurite outgrowth rates are significantly decreased compared with wild type. After SP600125 application, the retraction rate of jnk2^−/− neurites is decreased compared with jnk3^−/− and wild-type neurites, and no retraction of jnk1^−/−neurites occurs. B, Advancement or retraction of growth cones measured 90 min before and 90 min after SP600125 application. Within a few minutes after JNK inhibition, neurites from jnk2^−/− and jnk3^−/− neurons retract, whereas jnk1^−/− neurites continue growing during the remaining recording period. Response time for growth cones to collapse after SP600125 application: jnk2^−/− and jnk3^−/− growth cones collapse rapidly, in contrast to jnk1^−/− growth cones. D–F, Frames of time-lapse recordings illustrating the outgrowth and typical response of neurites to JNK inhibition from jnk1^−/−, jnk2^−/−, and jnk3^−/− DRG neurons, 90 min before and after SP600125 addition. Arrowheads, Growth cones. Scale bars, 50 μm. ***p < 0.001; **p < 0.01. Error bars indicate SEM.

Figure 5.

Scaffold protein JIP1 is required for neurite regeneration. A, B, and merge in C, JIP1 immunostaining is enriched in growth cones of regenerating DRG neurites, shafts of which are visualized by total MAP1B immunostaining. D, Western blot analysis of P-JNK in protein extracts from wild-type or jip1^−/− DRG neurons cultured 48 h before lysis; the expression of GAPDH was monitored in the lower part of the same membrane as loading control. Lack of JIP1 results in a reduction of the 46 kDa form of P-JNK. E–G, Tubulin immunostaining of DRG neurons cultured for 48 h derived from wild-type (E), jip1^−/− (F), and jip1^−/− neurons with additional SP600125 treatment (G). Note the abnormal “curly” morphology of neurites from jip1^−/− DRG exhibiting more terminal branching; additional SP600125 treatment has no effect on jip1^−/− neurites. H, Quantification of total neuritic length of wild-type versus jip1^−/− neurons reveals a difference of 70.4%; in contrast, additional SP600125 treatment has only a slight effect on neurite length of jip1^−/− DRG neurons (n ≥ 55). I, Diagram showing the distribution of total neuritic lengths of wild-type versus jip1^−/− DRG neurons, the latter cultured with or without additional SP600125 treatment. J, Mean speed of growth cone advancement from wild-type and JIP1-deficient DRG neurons showing that the outgrowth rate of jip1^−/− neurite is drastically decreased. K, Growth cone advancement or retraction measured 90 min before and 90 min after SP600125 application, showing that no neurite retraction occurs from jip1^−/− neurons during the remaining recording time, as illustrated in L, showing corresponding frames of time-lapse recordings. Arrowheads, Growth cones. Scale bars: A, C, 15 μm; E–G, 100 μm; L, 50 μm. ***p < 0.001; *p < 0.05. Error bars indicate SEM.

Figure 6.

Regulation of stathmin and MAP1B phosphorylation by JNKs: Western blot analysis with GADPH used as internal loading control. A, Relative amounts of total stathmin and stathmin phosphorylated on serine 25 (P-Ser25) in lysates of wild-type and JNK-deficient DRG neurons cultured for 48 h; note the decrease in stathmin phosphorylation in regenerating jnk1^−/− neurons. B, MAP1B staining of protein extracts from wild-type and JNK-deficient DRG neurons cultured for 48 h, showing a shift in the protein band representing MAP1B from higher to lower apparent molecular weight for jnk1^−/− and jnk2^−/−, but not jnk3^−/− neurons. This shift reflects a reduced level of MAP1B phosphorylation as demonstrated in C, MAP1B in lysates of N2A neuroblastoma cells cultured for 48 h is phosphorylated under control conditions (lane 1); experimental dephosphorylation by AP treatment of lysates (lanes 3, 4) results in the same shift to a lower molecular weight as JNK inhibition by SP600125 (SP) addition to the N2A cultures (lane 2).

Figure 7.

JNK inhibition affects MAP1B phosphorylation in regenerating neurons. A–C, In control DRG neurons, distal parts of neurites are enriched in MAP1B-P and characterized by low levels of detyrosinated tubulin (representing stable microtubules). D–F, Higher magnification of a growth cone showing the presence of MAP1B-P at the leading edge, where no colocalization with detyrosinated tubulin can be found. G–I, Double immunostaining reveals that total MAP1B is distributed in all parts of the neuron (green), whereas MAP1B-P (red) is mainly localized in neurites and exhibits a proximo-distal gradient. J, K, Application of SP600125, although not altering total MAP1B levels, dramatically decreases the neurite content in MAP1B-P, concomitantly with growth cone retraction (see also insets). L, M, Immunostaining for MAP1B-P, compared with total MAP1B, on regenerating jip1^−/− neurons reveals that lack of JIP strongly affects the level of MAP1B phosphorylation. Scale bars: A–C, 50 μm; D–F, 10 μm; G–M, 100 μm.

Figure 8.

JNK1 and JNK2 regulate MAP1B phosphorylation. Immunostaining for total MAP1B (left row) and MAP1B-P (middle row; the right row represents merge images) of regenerating neurons from wild-type (A–C) and JNK-deficient mice. MAP1B-P levels are strongly reduced in neurites elongating from jnk1^−/− (D–F) and jnk2^−/− (G–I) neurons, whereas both the level and the proximo-distal distribution of MAP1B-P in jnk3^−/− neurites (J–L) are similar to those of wild type. Scale bars, 50 μm.

Figure 9.

MAP1B is required for neurite retraction induced by JNK inhibition. A–D, map1b^−/− neurons after 48 h in culture, with or without SP600125 treatment, visualized by tubulin immunostaining. Note the increased number of branching points on the neurite tree of map1b^−/− neurons. JNK inhibition by SP600125 addition to the culture medium induces growth arrest and loss of growth cones (arrowheads), but no retraction bulbs are observed along the neurites, in contrast to wild-type neurons (compare Fig. 2G). E, F, Quantitative analysis reveals no significant differences in total neurite length and length of longest neurites between map1b^+/+ and map1b^−/− neurons under control conditions (gray bars); however, whereas SP600125 application (black bars) dramatically reduces total neurite length and length of longest neurites on map1b^+/+ neurons, JNK inhibition has only very limited effect on map1b^−/− neurites (n ≥ 52). G, Time-lapse recording illustrating the typical response of a map1b^−/− neurite: After SP600125 addition (at t = 60 min), growth cones collapse, neurites stop extending, but no retraction is observed (arrowheads) (see movie in supplemental data S6, available at www.jneurosci.org as supplemental material). Scale bars: A, C, 50 μm; B, D, 15 μm; G, 25 μm. ***p < 0.001. Error bars indicate SEM.

Figure 10.

Model of JNK functions in axonal regeneration. This model is based on previous studies (see Discussion) and incorporates our present findings and hypotheses. a, Axotomy provokes an activation of JNK signaling leading to phosphorylation of transcription factors (including c-jun, ATF3, and STAT3) that, in turn, will regulate the expression of regeneration-associated genes. During early stages of DRG neuron regeneration, specific activation of JNK2 and JNK3 may mediate transcriptional events required for neurite growth initiation in the “correct” time window. b, JNK activation is maintained during neurite extension. Signaling by P-JNK1 and -2, present in axon shaft and growth cone, regulates cytoplasmic effectors that finally influence microtubule (MT) dynamics. Among cytoplasmic effectors of JNKs are MT-destabilizing (stathmin) and -stabilizing proteins (MAP1B). Phosphorylation of stathmin (by JNK1) family members overcomes their destabilizing function on MT plus ends, whereas phosphorylation of MAP1B (by JNK1 and JNK2) affects its binding to MTs or bind more labile MTs. c, JNK inhibition induces retraction of previously formed axons. Nonphosphorylated stathmin sequesters tubulin dimers and thereby prevents MT polymerization and axon outgrowth. In parallel, a decrease in MAP1B phosphorylation will increase the binding of MAP1B to MTs, enhancing their stabilization and protecting them from depolymerization. Process retraction, crucial for correct axonal pathfinding, is based on two independent molecular mechanisms: Extension forces generated by the action of dynein on MTs are counterbalanced by actin–myosin contractility resulting in axon retraction. The dynamic interaction between the two opposed forces likely involves competition between MAP1B and dynein.