Enhancement of Peripheral Nerve Regrowth by the Purine Nucleoside Analog and Cell Cycle Inhibitor, Roscovitine

Abstract

Peripheral nerve regeneration is a slow process that can be associated with limited outcomes and thus a search for novel and effective therapy for peripheral nerve injury and disease is crucial. Here, we found that roscovitine, a synthetic purine nucleoside analog, enhances neurite outgrowth in neuronal-like PC12 cells. Furthermore, ex vivo analysis of pre-injured adult rat dorsal root ganglion (DRG) neurons showed that roscovitine enhances neurite regrowth in these cells. Likewise, in vivo transected sciatic nerves in rats locally perfused with roscovitine had augmented repopulation of new myelinated axons beyond the transection zone. By mass spectrometry, we found that roscovitine interacts with tubulin and actin. It interacts directly with tubulin and causes a dose-dependent induction of tubulin polymerization as well as enhances Guanosine-5'-triphosphate (GTP)-dependent tubulin polymerization. Conversely, roscovitine interacts indirectly with actin and counteracts the inhibitory effect of cyclin-dependent kinases 5 (Cdk5) on Actin-Related Proteins 2/3 (Arp2/3)-dependent actin polymerization, and thus, causes actin polymerization. Moreover, in the presence of neurotrophic factors such as nerve growth factor (NGF), roscovitine-enhanced neurite outgrowth is mediated by increased activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways. Since microtubule and F-actin dynamics are critical for axonal regrowth, the ability of roscovitine to activate the ERK1/2 and p38 MAPK pathways and support polymerization of tubulin and actin indicate a major role for this purine nucleoside analog in the promotion of axonal regeneration. Together, our findings demonstrate a therapeutic potential for the purine nucleoside analog, roscovitine, in peripheral nerve injury.

Keywords: axon; cytoskeleton; injury; peripheral nerve; regeneration.

Figure 1

Roscovitine enhances nerve growth factor (NGF)-mediated neurite outgrowth in PC12 cells. (A) PC12 cells were plated as described in “Materials and Methods” Section. After 18 h in low serum DM, cells were treated with roscovitine alone (10 μM, upper right panel), NGF alone (10 ng/ml NGF; lower left panel), or NGF and roscovitine simultaneously (lower right panel). The upper left panel shows cells that were not treated with either roscovitine or NGF. Photographs were taken 24 h after treatment. Scale bar = 50 μM. (B) Shows the number of cells with neurites longer than 20 μM. Measurements were taken from 50 cells in each treatment (n = 3). Statistical significance using student’s t-test was set at p < 0.05*. (C) Neurite lengths of differentiating PC12 cells were measured 24 h after treatment with NGF in the absence or presence of roscovitine at various concentrations. Untreated cells were used as control. Values are means ± SD of three (n = 3) independent experiments. Statistical significance using student’s t-test was set at p < 0.05*. (D) Viability of cells treated with various concentrations of roscovitine was assessed after 24 h by cell counting using a hemocytometer. Values are means ± SD of three (n = 3) independent experiments. Statistical significance using student’s t-test was set at p < 0.05*.

Figure 2

Roscovitine enhances axonal regrowth of pre-injured rat primary dorsal root ganglia (DRG) sensory neurons. (A) Neurons isolated from rat L4–L6 DRGs 3 days after sciatic nerve transection were cultured on laminin and poly-L-lysine coated cover slips in primary neuron media, containing N-2 supplement, BSA and 0.2 ng/ml NGF, in the absence or presence of roscovitine (0.2, 2.0 or 10 μM). After 48 h, cells were fixed in 4% paraformaldehyde and immunostained with β-III tubulin antibody. Scale bar = 50 μM. (B) Axon lengths of DRG neurons exposed to different concentrations of roscovitine. Values are means ± SD of three independent experiments. Statistical significance using student’s t-test was set at p < 0.05*.

Figure 3

Roscovitine increases the number of myelinated axons in regenerating rat sciatic nerve. Adult male SD rats were subjected to sciatic nerve transection as described in “Materials and Methods” Section. (A) A schematic diagram showing the sciatic nerve lesion, the regeneration chamber and cutting site of EPON embedded section that lies perpendicular to the direction of the bridge. (B) Analysis of the number of axons with Schwann cells (SC) in control and roscovitine-treated rats 7 days after sciatic nerve transection. Axonal outgrowth were measured by serial counts (fields 1–7) proximal to the distal stump of a cut sciatic nerve by co-immunohistochemical staining for neurofilament (for neurons) and glial fibrillary acid protein (GFAP; for SC). Values are means ± SD; n = 4. Changes in values did not reach to statistical significance using Student’s t-test at p ≤ 0.05. (C) Representative image of axons with SC (examined in B) in control and roscovitine-treated rats at 7 days. Arrowheads are directed at axons. (D) Twenty-one days post-transection, axonal regeneration was evaluated by morphometric analysis of toluidine blue-stained EPON embedded cross sections of sciatic nerve bridges treated with Ringer’s solution (left panel) or 10 μM roscovitine (right panel). Arrow is directed at a myelinated axon. (E) Analysis of the number of myelinated axons (left panel) and axon caliber (right panel) in control (Ringer’s solution-treated) and roscovitine-treated rats 21 days after sciatic nerve transection. Values are means ± SD; n = 5. Statistical significance using student’s t-test was set at p < 0.05*.

Figure 4

Identification of roscovitine interacting proteins in differentiating PC12 cells. (A) Neuronal differentiation of PC12 cells was induced using NGF. After pre-clearing with agarose beads, differentiating PC12 cell lysates were subjected to pull-down assay using roscovitine-agarose beads. The pulled-down proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Sypro-Ruby Red (upper panel). A parallel SDSPAGE loaded with the same samples and analyzed by western blotting showed immunoreactivity to extracellular signal-regulated kinases 1/2 (ERK1/2) and Cdk5. (B) Pulled-down proteins were also analyzed by mass spectrometry. A list of roscovitine interacting proteins in differentiating PC12 cells treated with NGF and roscovitine is shown.

Figure 5

Roscovitine interacts with tubulin directly, and enhances tubulin polymerization. (A) Mouse brain homogenate (lane 1) and purified tubulin (lanes 3 and 4) were pre-cleared/-incubated with agarose beads and subsequently incubated with roscovitine-agarose beads. Tubulin binding to roscovitine beads was analyzed by western blotting using a tubulin antibody. Purified tubulin served as positive control (lane 2). For negative control (lane 3), tubulin was pre-incubated with 0.2 mM roscovitine (ros) prior to incubation with roscovitine-agarose beads. Note that no agarose-tubulin binding was observed after a second pre-clearing of brain homogenates. (B) Spectrophotometric tubulin polymerization assay was performed using a kit, and with slight modification of the manufacturer’s (Cytokeleton Inc.) protocol. To obtain detectable levels of polymerization, the recommendation for the in vitro tubulin polymerization assay kit is to use tubulin at 2 mg/ml in fluorescence buffer (80 mM PIPES pH 6.9, 2.0 mM MgCl₂, 0.5 mM EGTA, 10 μM fluorescent reporter) + 1 mM Guanosine-5′-triphosphate (GTP). As R-roscovitine is a synthetic GTP analog, a comparable amount of R-roscovitine was used: 0.2 mM or 0.4 mM when used alone or 0.4 mM when used together with 0.5 mM GTP. Analysis of: polymerization of 4′,6-diamidino-2-phenylindole (DAPI)-tubulin alone (□) or (1) incubated with increasing concentrations of roscovitine (°; 0.2 mM or 0.4 mM); or (2) GTP (•, positive control; 0.5 mM), or GTP and roscovitine (Δ; 0.5 mM and 0.4 mM, respectively). Data from the two independent analyses with similar range of fluorescence values are shown together.

Figure 6

Roscovitine interacts with actin indirectly, and enhances Actin-Related Proteins 2/3 (Arp2/3)/Wave1-mediated actin polymerization. (A) Mouse brain homogenate (lane 1) and purified actin (lanes 3 and 4) were pre-cleared/-incubated with agarose beads and subsequently incubated with roscovitine-agarose beads. Actin binding to roscovitine beads was analyzed by western blotting using an actin antibody. Purified actin served as positive control (lane 2). For negative control (lane 3), actin was pre-incubated with 0.2 mM roscovitine (ros) prior to incubation with roscovitine-agarose beads. The lack of actin immunoreactivity in lane 4 indicates that actin in the brain homogenate (lane 1) interacts with roscovitine indirectly. Note that no agarose-actin binding was observed after a second pre-clearing of brain homogenates. (B) SDS-PAGE and Coomassie brilliant blue staining of purified Glutathione S-transferase-cyclin-dependent kinases 5 (GST-Cdk5; left panel), GST-p25 (middle panel; Cdk5 activator) and Strep-Wave1 complex (right panel). Asterisks correspond to the heat shock chaperon protein that copurifies with GST-Cdk5 and GST-p25. (C) Polymerization curve of pyrene-actin incubated with Arp2/3 ± Wave1 complex ± Cdk5/p25 ± preincubation with roscovitine. Values shown are obtained following a preincubation time of 11 min and fluorescence was set to an arbitrary value of zero. For all treatments, fluorescence start to plateau at ~27 min. (D) Western blot for Wave1 phospho-Ser310 showing that Cdk5/p25 phosphorylates Wave1 at Ser310 and this phosphorylation is inhibited by roscovitine. (E) Densitometric analysis of the representative blot in (D) using the NIH Image-J 1.61 software.

Figure 7

Roscovitine enhances the activation of ERK1/2 and p38 mitogen-activated protein kinase (MAPK) in differentiating NGF-treated PC12 cells. (A) Representative set of blots (from three sets/independent experiments) of lysates of PC12 cells plated and treated as described in “Materials and Methods” Section. After serum starvation for 18 h, cells were treated with NGF alone (10 ng/ml; left panel), roscovitine alone (10 μM; right panel) or NGF+roscovitine simultaneously (middle panel), and lysed at 0, 1, 3, 10, 30, 90 and 180 min post-treatment. Equal amounts of protein samples were then resolved in 12.5% SDS-PAGE and subjected to western blot analysis using phospho-antibodies against ERK1/2, p38 MAPK and AKT. Additional test for loading equivalent amounts of protein was performed by Ponceau S staining. (B) The ratios of the band intensities [from the representative blots in (A)] of phospho-ERK1/2, phospho-p38 MAPK and phospho-Akt vs. total ERK, total p38 MAPK and total Akt, respectively, were determined following densitometric scanning of bands using the NIH Image-J 1.61 software. Note the different Y-axis scales for p-ERK1/total ERK (left) and p-ERK2/total ERK (right). Analysis of the ratios of the band intensities of phospho-AKT vs. total AKT did not show any considerable changes at different time points in the three treatment groups. (C) Proposed molecular mechanisms by which roscovitine enhances NGF-induced axonal regrowth. Roscovitine promotes axonal regrowth by inducing tubulin and actin polymerization (left panel). In the presence of NGF, roscovitine enhances axonal regrowth through activation of the ERK1/2 and p38 MAPK pathways.