Inactivation of glycogen synthase kinase 3 promotes axonal growth and recovery in the CNS

Abstract

Axonal regeneration is minimal after CNS injuries in adult mammals and medical treatments to recover neurological deficits caused by axon disconnection are extremely limited. The failure of axonal elongation is principally attributed to the nonpermissive environment and reduced intrinsic growth capacity. In this report, we studied the role of glycogen synthase kinase-3 (GSK-3) inactivation on neurite and axon growth from adult neurons via combined in vitro and in vivo approaches. We found that the major CNS inhibiting substrates including chondroitin sulfate proteoglycans could inactivate protein kinase B (Akt) and activate GSK-3beta signals in neurons. GSK-3 inactivation with pharmacologic inhibitors enhances neurite outgrowth of dorsal root ganglion neurons derived from adult mice or cerebellar granule neurons from postnatal rodents cultured on CNS inhibitors. Application of GSK-3 inhibitors stimulates axon formation and elongation of mature neurons whether in presence or absence of inhibitory substrates. Systemic application of the GSK-3 inhibitor lithium to spinal cord-lesioned rats suppresses the activity of this kinase around lesion. Treatments with GSK-3 inhibitors including a clinical dose of lithium to rats with thoracic spinal cord transection or contusion injuries induce significant descending corticospinal and serotonergic axon sprouting in caudal spinal cord and promote locomotor functional recovery. Our studies suggest that GSK-3 signal is an important therapeutic target for promoting functional recovery of adult CNS injuries and that administration of GSK-3 inhibitors may facilitate the development of an effective treatment to white matter injuries including spinal cord trauma given the wide use of lithium in humans.

Figures

Figure 1.

Axon growth inhibitors transiently alter the activities of Akt and GSK-3β signals in cultured neuronal cells. A, B, Western blot indicates the levels of phosphorylated Akt (Akt-p, active form) and GSK-3β (GSK-p, inactive form) in the lysates of PC12 cells 24 h after differentiation with NGF (50 ng/ml). Application of inhibitor CSPGs (CS, 1.5 μg/ml) significantly reduced the levels of Akt-p (A) and GSK-p (B) 2–5 min after exposures, although the total protein levels (Akt-t, GSK-t) are similar in these lysates. The graphs at bottom indicate the level of Akt-p and GSK-p in PC12 lysates quantified from several separate experiments. C, D, Western blot indicates the levels of Akt-p and GSK-p in the lysates of CGNs 24 h after cultures derived from P7–P9 mice. Similarly, CSPGs (1.5 μg/ml) significantly attenuated the levels of Akt-p (C) and GSK-p (D) a few minutes after exposures. E, F, The levels of Akt-p and GSK-p in the lysates of PC12 cells 24 h after differentiation with NGF (50 ng/ml) were determined from PC12 cell lysates after application of myelin inhibitor MAG (570 ng/ml). MAG moderately reduced the levels of Akt-p (E), although it did not significantly affect levels of GSK-p after exposure. The bar graphs indicate means ± SEM from four to six separate experiments. The differences indicated are compared with the PBS-treated controls without CSPGs or MAG (*p < 0.05, **p < 0.01, Student's t test). CS, CSPG.

Figure 2.

GSK-3 inhibitors stimulate neurite outgrowth from cultured DRG neurons or CGNs on CNS myelin or CSPG substrates. A, The representative examples of dissociated DRG neurons from 7–10 week mice were treated with lithium (Li; 3 mm), SB (7.5 μm), or vehicles (Veh) starting after cell plating on PBS, myelin (Mye; 50 μg/ml), or CSPG (1.5 μg/ml) substrates. Cells were fixed and stained with rhodamine phalloidin 24 h after growth. Lithium and SB415286 stimulated neurite growth on myelin or CSPGs. B, C, The neurite outgrowth per DRG neuron was manually traced and quantified 24 h after cell plating on CNS myelin (B) or CSPG (C) substrates using NIH and Photoshop software. The bar graphs indicate means ± SEM from five to nine separate experiments (30–80 representative neurons were analyzed from 7 images in each experiment). The differences indicated are compared with the vehicle-treated controls. Treatments with Li or SB significantly increased neurite length of DRG neurons on CNS myelin and CSPGs 24 h after cell plating. D, The representative examples of dissociated CGNs derived from P7–P9 mice were treated with lithium (3 mm), SB415286 (7.5 μm), or vehicles starting after cell plating on PBS, myelin (50 μg/ml), or CSPG (1.5 μg/ml) substrates. The cells were fixed and stained with rhodamine phalloidin 24 h after growth. E, F, The neurite outgrowth per neuron was quantified 24 h after cell plating on CNS myelin (E) or CSPGs (F). The numbers in bar graphs indicate means ± SEM from six to eight separate experiments (100–150 representative neurons were analyzed from 7 images in each experiment). The differences indicated are compared with the vehicle-treated controls (*p < 0.05, **p < 0.01, Student's t test). Treatments with Li or SB significantly increased neurite length of CGNs cultured on CNS myelin and CSPGs. Scale bars, 50 μm.

Figure 3.

GSK-3β inhibitors promote axonal formation and enhance axonal length in DRG neuronal cultures derived from adult mice. A, The representative examples of DRG neurons with axons cultured from 8 wk C57BL/6 mice 4 d after cell plating. The neurons were treated with vehicle (Veh; saline), lithium (Li; 3 mm), or SB (7.5 μm) in absence (top row) or presence (bottom row) of purified CNS myelin (20 μg/ml). After fixation, the cells were stained with axonal marker neurofilament (arrows). B–D, Bar graphs indicate the percentage of axon-forming neurons (B), the length of axons in each neuron with axons (C), and branching numbers in each axon (D) of DRG cultures without exposure to CNS myelin axon inhibitors. E–G, Bar graphs indicate the percentage of axon-forming neurons (E), the length of axons in each axon-forming neuron (F), and branching numbers of each axon (G) in DRG cultures in presence of CNS myelin. The means ± SEM are reported from five to seven separate experiments. Multiple images (7–8) were collected and quantified from an individual experiment in each group (*p < 0.05, **p < 0.01 compared with vehicle-treated cultures, Student's t test). Treatments with lithium (3 mm) or SB (7.5 μm) significantly enhanced the number of axon-forming neurons and the length of axons in axon-bearing neurons on CNS myelin (E, F). H, Synaptic marker synaptophysin was visualized via immunostaining in DRG neurons derived from postnatal 2 d mice 5 d after cell plating. GSK-3 inhibitors enhanced the expression of synaptic structural protein in neuronal bodies (arrows) as well as in axonal cylinders (arrowheads). Scale bars, 50 μm.

Figure 4.

Traumatic injury attenuated Akt activity and treatment with lithium (Li) suppressed GSK-3β activity by enhancing GSK-p level in the lesioned rat spinal cord. A, B, Akt-p and GSK-p were analyzed from the tissue lysates of fresh spinal cord 3 mm rostral to and 3 mm caudal to a lesion 24 h after injury at T8 (or the same level of spinal cord in no-SCI controls) via Western blot with antibody specific to phosphorylated Akt at Ser473 (active) and GSK-3β at Ser-9 (inactive). Total Akt and GSK-3β from the same tissue samples were detected via immunoblotting (bottom bands). Bar graph below bands indicates the densitometric analysis of Akt-p and GSK-p levels in the lesioned spinal cord (means ± SEM; n = 4 from 4 rats in each group). C, The levels of Akt-p and GSK-p were examined from the fixed sagittal sections of spinal cord white matter (2–4 mm from lesion) at midthoracic level from three groups of rats 24 h after SCI. Both Akt-p and GSK-p were visualized via immunostaining against these proteins. The signal for Akt-p was attenuated in spinal cord around lesion compared with noninjury controls at the same spinal cord level. Lithium treatment enhanced GSK-p signal. Scale bar, 50 μm. D, E, Akt-p and GSK-p signals were quantified from white matter of spinal cord sections 2–4 mm rostral to and caudal to lesion (means ± SEM; n = 5–7 from 5–7 images in each group; *p < 0.05, **p < 0.01 compared with non-SCI for Akt-p or with SCI controls for GSK-p, Student's t test).

Figure 5.

Systemic application of lithium stimulates corticospinal fiber sprouting rostral to a unilateral transection in mouse spinal cord. A, Schematic of transverse spinal cord illustrates the transected area of cord in mice. The small arrow indicates the dorsal CST area labeled with BDA. B–E, BDA-labeled dorsal CST axons are visualized in transverse sections 5–7 mm rostral to SCI in vehicle- (B, C) or lithium (Li)-treated (D, E) animals. A few BDA-labeled sprouts (arrowheads) project into the gray matter in controls (dorsal is up). Treatment with lithium for 4 weeks exhibits a higher density of CST sprouts extending laterally into gray matter 4 weeks after SCI. Scale bars: A, 200 μm; (in B) B, D, 50 μm; (in B) C, E, 25 μm.

Figure 6.

Systemic application of GSK-3 inhibitors promotes CST axonal growth around lesion in rats with dorsal transection or contusion injuries. A, C, Sagittal sections in SCI controls show no regenerative CST growth in rostral and caudal spinal cord 6 weeks after a dorsal transection injury at T7. B, Schematic drawing illustrates transverse spinal cord and transected area (shaded) in rat dorsal transection model. D, Similar section from a lithium (Li)-treated rat indicates a number of CST fiber sprouts rostral and caudal to the transection lesion, particularly in the gray matter (GM) areas (the dorsal is up in all of these sections). E, F, Higher-magnification images from D demonstrate the meandering course of CST sprouting fibers (arrowheads) around lesion. G, Quantification of CST fibers ≥100 μm in fiber length or in longest axis of sprouting complex outside of ventral CST is illustrated at various distances from longitudinal sections caudal to the transection lesion (mean ± SEM; n = 7, 9 rats in the control and lithium groups; *p < 0.05, Student's t test). H, Transverse sections of spinal cord 11–15 mm caudal to the transection lesion from vehicle-treated rats indicated a few spared ventral CST axons (arrowhead), but few BDA-labeled CST fibers were found in the dorsal part of spinal cord. Transverse sections of spinal cord at same level from lithium-treated rats displayed tracer-labeled CST axons in both ventral and dorsal part of spinal cord (arrowheads). I, Counting of BDA-labeled CST fibers from transverse sections 11–15 mm caudal to the transection lesion indicates more BDA-labeled fibers in the lithium group (mean + SEM; n = 7, 9 rats in the control and lithium groups, *p < 0.05, Student's t test). J, A parasagittal section containing the injury site (arrow) from a lithium-treated rat demonstrates the lesion area after a contusion injury. A large cavity is visualized in the lesion epicenter of the spinal cord. K, L, Bar graphs indicate quantification of CST fibers ≥100 μm in sprouting length from longitudinal sections 1–10 mm caudal to a moderate contusion lesion 8 weeks after injury in the vehicle- lithium-, and SB-treated rats. Treatments with lithium or SB significantly increase the CST fiber number caudal to the contusion. WM, White matter. M, Counting of BDA-labeled CST fibers from transverse sections 11–15 mm caudal to the contusion lesion displays more BDA-labeled fibers in the lithium or SB group 8 weeks after SCI (mean ± SEM; n = 10, 9 and 9 rats with contusion SCI in three groups, respectively; *p < 0.05, **p < 0.01, Student's t test). Scale bars: (in A) A, D, J, 500 μm; B, 200 μm; (in C) C, E, F, 100 μm; H, 20 μm.

Figure 7.

Systemic GSK-3 inhibitors enhance serotonergic fibers in the spinal cord 11–15 mm caudal to a dorsal transection or a contusion injury in rats. A, B, Transverse sections of central and ventral spinal cord 11–15 mm rostral to SCI reveal the high density of serotonin fibers (arrows) in SCI-control rats. C–F, Transverse sections of spinal cord 11–15 mm caudal to the lesion displayed the reduced 5-HT fibers (arrows) 6 weeks after the transection injury at T7 (Trans-SCI), but treatment with lithium increased serotonergic fibers (arrows) in both central and ventral part of spinal cord (E, F) compared with those from vehicle-treated rats (C, D). G, Serotonin fiber length was measured from gray and white matter in dorsocentral areas and from gray matter in ventral horn of the spinal cord 11–15 mm caudal to transection lesion. The means ± SEM from 7 and 9 rats in control and lithium groups are reported. H–M, Transverse sections of spinal cord 11–15 mm caudal to the lesion displayed the reduced 5-HT fibers (arrows) 8 weeks after a contusion injury at T8 (Contu-SCI). Treatments with lithium or SB increased serotonergic fibers (arrows) in both central and ventral part of the spinal cord (J–M) compared with those from vehicle-treated rats (H, I). Scale bars: A (for A, C, E, H, J, L), 50 μm; B (for B, D, F, I, K, M), 50 μm. N, Serotonin fiber length was measured in dorsocentral and ventral areas of spinal cord 11–15 mm caudal to the contusion lesion. The means ± SEM from 10, 9 and 9 rats with contusion SCI in control, lithium, and SB groups are reported. **p < 0.01, Student's t test.

Figure 8.

Treatments with GSK-3 inhibitors improve the behavioral recovery after a dorsal transection or a moderate contusion injury in rats. A, Graph indicates the locomotor BBB score in the vehicle or lithium-treated rats after a dorsal transection injury (n = 7, 9 rats in the control and lithium groups; **p < 0.01, Student's t test). B, The examples of representative footprints from control (top) and lithium (Li, bottom) animals after a dorsal transection injury. The double-end arrows indicate the stride distances measured from the footprints. C, Footprint analysis revealed a greater stride length and a shorter stride width in lithium-treated animals than in controls. The grid-walk and footprint tests were performed 6 weeks after the dorsal transection injury. D, E, Graphs indicate BBB score (D) and grid-walking errors (E) in the contusion-injured rats treated with vehicle, lithium, or SB. F, The examples of representative footprints from control (top), lithium (middle), and SB (bottom) animals after a contusion injury. The double-ended arrows illustrate the stride width measured from the footprints. G, Footprint analysis revealed a shorter stride width in lithium- or SB-treated animals than in the controls. The grid-walk and footprint tests were performed at 8 weeks after the contusion. Means ± SEM are reported. The differences indicated are compared with the saline-treated group (*p < 0.05, **p < 0.01, Student's t test).