Spy1 Protein Mediates Phosphorylation and Degradation of SCG10 Protein in Axonal Degeneration*

Background: SCG10 is a novel axonal maintenance factor, and rapid SCG10 loss after injury requires JNK activity. Results: Spy1 mediated SCG10 phosphorylation and degradation, partly in a JNK-dependent manner, and regulated injury-induced axonal degeneration. Conclusion: Spy1 is an important regulator of SCG10 and axon degeneration. Significance: Spy1 may be a novel axo-protective therapeutic target for axon loss. Axon loss is a destructive consequence of a wide range of neurological diseases without a clearly defined mechanism. Recent data demonstrate that SCG10 is a novel axonal maintenance factor and that rapid SCG10 loss after injury requires JNK activity; how JNK induces degradation of SCG10 is not well known. Here we showed that SCG10 was a binding partner of Spy1, a Speedy/RINGO family protein, which participated in cellular response to sciatic nerve injury. During the early stage of axonal injury, Spy1 expression was inversely correlated with SCG10. Spy1 mediated SCG10 phosphorylation and degradation partly in a JNK-dependent manner. Inhibition of Spy1 attenuated SCG10 phosphorylation and delayed injury-induced axonal degeneration. Taken together, these data suggest that Spy1 is an important regulator of SCG10 and can be targeted in future axo-protective therapeutics.

Axonal degeneration is a major cause of neurological disabilities, which include hereditary neuropathies, glaucoma, diabetic neuropathy, and Alzheimer and Parkinson disease (1,2). Although the precise mechanism of axon loss is poorly understood, it is clear that axons are dismantled by a carefully orchestrated mechanism. Proteasome activity promotes axon breakdown (3), potentially via the degradation of factors that are required for axonal maintenance after injury, such as the Wallerian degeneration slow (Wld S ) fusion protein or its enzymatically active component nicotinamide nucleotide adenylyltransferase (NMNAT) (4 -9). On the other hand, additional maintenance factors may function in the injured axons to establish a set point at which injury-induced degeneration pathways are engaged. Therapies targeting the axonal degeneration process itself are notably absent. Hence, elucidating the mechanism of axonal degeneration may help develop novel axo-protective therapeutic strategies.
Superior cervical ganglion 10 (SCG10), also known as stathmin 2 (STMN2), is a neuronally expressed stathmin family protein that regulates microtubule dynamics and protein trafficking (10,11). SCG10 is highly expressed during development and plays an important role in axonal outgrowth by modulating microtubule stability (12,13). Interestingly, recent data have demonstrated that SCG10 is a novel axonal maintenance factor whose loss is permissive for injury-induced axonal degeneration. Experimental depletion of SCG10 results in accelerated degeneration of injured axons, and enforced maintenance of SCG10 levels in axons following injury is sufficient to delay degeneration (14). Although previous data showed that SCG10 was an axonal JNK substrate (13) and that it rapidly degraded during injury-induced axonal degeneration in a JNK-dependent manner (14), how JNK could induce degradation of SCG10 is not well known.
Spy1 was first identified as an activator of CDKs, 3 although the members have no homology to cyclin proteins (15,16). Spy1 enhances cell proliferation, promotes the G 1 /S transition (17), and inhibits apoptosis in response to UV irradiation (18). Spy1 levels are up-regulated in variety of tumor tissues (19,20); overexpression of Spy1 accelerates tumorigenesis (21), and knockdown of Spy1 can reduce breast cancer cell growth in vivo. In addition to its role in cell cycle regulation, Spy1 is implicated in mammary development (21). Our previous data showed that Spy1 was ubiquitously expressed in the lumbar spinal cord, including neurons and glial cells (22), and participated in the pathological process in response to sciatic nerve injury (23).
To tackle the possible role of Spy1 in the central nervous system, we performed a yeast two-hybrid screening using a human fetal brain complementary DNA library. Our data demonstrate that Spy1 directly binds to SCG10 and modulates its phosphorylation and stability, delaying injury-induced axonal degeneration partly in a JNK-dependent manner. Therefore, This article has been withdrawn by the authors. The authors mistakenly uploaded images from Shin, J. E., Miller, B. R., Babetto, E., Cho, Y., Sasaki, Y., Qayum, S., Russler, E. V., Cavalli, V., Milbrandt, J., and DiAntonio, A. (2012) SCG10 is a JNK target in the axonal degeneration pathway. Proc. Natl. Acad.
Sci. U.S.A. 109, E3696-E3705 to Fig. 4D. The authors offer their sincere apologies for their carelessness and failure to scrupulously preserve the original data.
Spy1 is a novel regulator of axon degeneration and as such positioning itself as a potential therapeutic target.

Materials and Methods
Expression Plasmid Preparation-SCG10 was isolated by PCR from a human fetal brain complementary DNA library and inserted into pcDNA3.1-Myc vector. The identical method was used for GST-SCG10, His-Spy1, HA-Spy1, and their mutants. SCG10 phosphorylation site mutants S50A, S62A, S73A, and S97A were created from wild-type SCG10. All constructs were verified by sequencing. GFP-tagged lentivirus sh-Spy1 was purchased from GeneChem (Shanghai, China).
Cell Culture and Transfection-HEK-293 cells were maintained in Dulbecco's modified Eagle's medium (Gibco). Medium was supplemented with 10% fetal bovine serum and cultured at 37°C in a humidified incubator at 5% CO 2 . Cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
Dorsal root ganglions (DRGs) from embryonic day 17.5-18.5 rat (Charles River) were cultured in poly-D-lysine-(Sigma) and laminin-(Sigma) coated 24-well plates (Corning). Collected DRGs were trypsinized for 20 min at 37°C, triturated in medium, and plated as a spot at a density of approximately two DRGs per well in 2 l of medium. The plates were incubated for 20 min at 37°C to attach cells to the plastic followed by the addition of Neurobasal medium (Invitrogen) supplemented with 2% (v/v) B27 (Invitrogen), 25 ng/ml nerve growth factor (Promega), and 1 M 5-fluoro-2Ј-deoxyuridine and 1 M uridine (Sigma) to block cell division of nonneuronal cells. Cultures were maintained for 8 -9 days before axotomy or drug treatment. We axotomized cultures of DRG neurons with a FIGURE 1. SCG10 is a binding partner of Spy1. A, Spy1 interacts with SCG10 in vitro. The GST pulldown assay was performed by incubating in vitro-translated His-tagged Spy1 with purified GST-SCG10 immobilized on glutathione-Sepharose beads. GST-SCG10 but not GST was shown to pull down Spy1. Arrows indicate GST and GST-SCG10 bands, and Coomassie Blue staining indicates the loading amounts. WB, Western blot. B, HA-tagged Spy and Myc-tagged SCG10 were coexpressed in HEK-293 cells, and they could pull down each other. IP, immunoprecipitation. C, the interaction between endogenous Spy1 and SCG10 in DRG neurons, analyzed by immunoprecipitation using a SCG10-specific antibody. Myc-tagged SCG10 mutants and HA-tagged Spy mutants were co-expressed in HEK-293 cells for immunoprecipitation assays. D and E, the stathmin-like domain (SLD) of SCG10 was required for binding to Spy1. F and G, the Speedy/RINGO domain (S/R) of Spy1 was required for binding to SCG10. microscalpel. In addition, we added lentivirus to the culture at 4 -5 days and allowed 4 -5 days for expression. To confirm efficient overexpression or knockdown of a gene, we monitored fluorescence from Venus and performed Western blot analysis to assess the level of the protein of interest.
Western Blot Analysis-Cells were washed and collected from plates in PBS solution, resuspended with 2ϫ sample buffer, and boiled for 5 min. Proteins were then resolved in an 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore).
Rat in Vivo Sciatic Nerve Transection-We used adult Sprague-Dawley rats weighing 220 -250 g (Department of Animal Center, Nantong University). We anesthetized animals, made a small incision unilaterally to expose the sciatic nerve, transected the sciatic nerve with surgical scissors, and then sutured the incision. Three hours after transection, rats were euthanized by CO 2 , and the sciatic nerves were removed for Western blot analysis. From the transected nerve, only distal segments were collected. Rat experiments were performed under the supervision of Division of Comparative Medicine at Nantong University.
Phase-contrast Imaging and Quantifying Axon Degeneration in Vitro-Axon degeneration was analyzed as described previously (14). Briefly, phase-contrast images were obtained on an inverted light microscope (CKX41; Olympus) with a 20ϫ objective. Three nonoverlapping images of each well were taken at each time point and assessed for axon degeneration. Images were processed with the auto-level function in Photoshop (Adobe) for brightness adjustment. We then analyzed the images by using a macro written in ImageJ to calculate the degeneration index (26,27). After images were binarized, the total axon area was defined by the total number of detected pixels. The area of degenerated axon fragments was calculated using the particle analyzer function. To calculate the degeneration index, we divided the area covered by axon fragments by the total axon area. We averaged the indices of three images taken from the same well to calculate the mean degeneration index for each well. Degeneration studies were performed in at least three independent experiments.
Statistical Analysis-Values were expressed as mean Ϯ S.E. Student's t test was used to measure significance of differences between two groups. Statistical significance was defined as p Ͻ 0.05.

Results
SCG10 Is a Binding Partner of Spy1-To identify novel Spy1binding partners, we performed yeast two-hybrid screening using a human fetal brain complementary DNA library with Spy1 as bait and identified SCG10 as a putative binding protein (Table 1). To verify the potential interaction obtained with the yeast two-hybrid screening, we performed an in vitro GST binding assay and showed that in vitro-translated Spy1 interacted with GST-SCG10 but not with GST alone (Fig. 1A). Immunoprecipitation assays were then undertaken to test the intracellular interaction between Spy1 and SCG10. HA-tagged Spy and Myc-tagged SCG10 were co-expressed in HEK-293 cells, and they could pull down each other (Fig. 1B). Importantly, immunoprecipitation of endogenous SCG10 from DRG neurons using a SCG10-specific antibody also pulled down Spy1 protein (Fig. 1C).
To determine the protein domains required for the interaction between Spy1 and SCG10, Myc-tagged SCG10 mutants and HA-tagged Spy mutants were co-expressed in HEK-293 cells for immunoprecipitation assays. The data suggested that the stathmin-like domain of SCG10 was required for binding to FIGURE 2. Spy1 expression inversely correlates with SCG10 during the early stage of axonal injury. A, immunoblot analysis of endogenous SCG10 and Spy1 in cultured DRG axons with or without axotomy. Immunoblot against neuron-specific ␤3-tubulin confirms comparable amounts of protein loaded. B, the ratio of Spy1 or SCG10 protein relative to ␤3-tubulin in the uncut and cut axons by densitometry. The data are mean Ϯ S.E. ( n ϭ 3, *, #, p Ͻ 0.01, significantly different from the uncut group). C, immunoblot analysis of endogenous SCG10 and Spy1 in distal sciatic nerve with or without axotomy. Immunoblot against neuron-specific ␤3-tubulin confirms comparable amounts of protein loaded. D, the ratio of Spy1 or SCG10 protein relative to ␤3-tubulin in the uncut and cut axons by densitometry. The data are mean Ϯ S.E. ( n ϭ 3, *, #, p Ͻ 0.01, significantly different from the uncut group). Spy1 (Fig. 1, D and E) and that the Speedy/RINGO domain of Spy1 was required for binding to SCG10 (Fig. 1, F and G).
Spy1 Expression Inversely Correlates with SCG10 during the Early Stage of Axonal Injury-Previous data showed that SCG10 was a novel axonal maintenance factor (14). Given that our recent data showed that Spy1 participated in the pathological process response to sciatic nerve injury (23), we hypothesized that Spy1 might modulate SCG10 activity and participate in axon regeneration/degeneration. We first examined the expression of both proteins in an in vitro model of axonal injury. In DRG neurons, SCG10 expression was lost rapidly from injured distal axons during the first 3 h after axotomy (Fig. 2, A  and B), which was in agreement with previous studies (14,25). Interestingly, Spy1 level was increased in injured distal axons during the same period (Fig. 2, A and B). To further confirm these results, we performed an in vivo model of axonal injury of sciatic nerve. Indeed, SCG10 level decreased rapidly in injured distal sciatic nerves at the early stage after transection (Fig. 2, C  and D), whereas Spy1 level was increased in parallel (Fig. 2, C  and D). Therefore, our data suggested that Spy1 expression was inversely correlated with SCG10 during the early stage of axonal injury.
Spy1 Mediates SCG10 Phosphorylation and Degradation Partly in a JNK-dependent Manner-As Spy1 levels showed an inverse correlation with SCG10 after axonal injury, we speculated whether Spy1 could regulate SCG10 level. Forced expression of ectopic Spy1 resulted in a decrease of SCG10 in a dosedependent manner (Fig. 3, A and B). In contrast, treatment with the proteasome inhibitor MG132 blocked the loss of SCG10 (Fig. 3, C and D), indicating that Spy1 might impact SCG10 stability rather than its de novo synthesis Previous data showed that JNK phosphorylation of SCG10 targeted SCG10 for degradation (14). We then set out to deter-mine whether Spy1 might affect SCG10 phosphorylation in a JNK-dependent manner. Consistent with the observation that Spy1 promoted SCG10 degradation, it also enhanced SCG10 phosphorylation (Fig. 3E). To further identify the specific phosphorylation site(s) of SCG10 by Spy1, four candidate phosphor- ylation sites (serine 50, 62, 73 and 97) were individually substituted with alanine (termed S50A, S62A, S73A, and S97A). As shown in Fig. 3C, SCG10 mutant containing an alanine substitution at serine 62 instead of 50, 73, or 97 abolished Spy1-mediated phosphorylation, indicating that Spy1 specifically induced phosphorylation of SCG10 protein at serine 62.

Spy1 Mediates Axonal Degeneration
Spy1 induced SCG10 phosphorylation was apparently dependent on JNK kinase because treatment with a selective JNK kinaseinhibitorSP600125partlyabolishedSpy1-mediatedphosphorylation of SCG10 (Fig. 3F). Previous data showed that Spy1 could activate CDK2 (26), so we asked whether Spy1-mediated phosphorylation of SCG10 might occur downstream of CDK2. Our results showed that inhibition of CDK2 had no effect on Spy1-mediated phosphorylation of SCG10 (Fig. 3F). To further investigate the mechanism of Spy1-mediated phosphorylation of SCG10 on JNK kinase, we examined the effect of Spy1 on JNK activity. Our results showed that Spy1 increased JNK kinase activity, whereas SP600125 or TAT-TI-JIP153-163 inhibited it (Fig. 3G). Together, these data suggest that Spy1 mediates SCG10 phosphorylation and degradation partly in a JNK-dependent manner.
Inhibition of Spy1 Attenuates SCG10 Phosphorylation and Delays Injury-induced Axonal Degeneration-Next, we investigated the role of Spy1 in the degeneration of DRG neurons after axotomy. GFP-tagged lentivirus targeting Spy1 was used to infect DRG neurons. Spy1 shRNA #3 resulted in ϳ80% reduction in Spy1 level when compared with control shRNA (Fig.  4A). Previous data showed that phosphorylated SCG10 appeared at a higher molecular weight than nonphosphorylated SCG10 (13). Our results showed that Spy1 knockdown preferentially preserved lower molecular weight SCG10 species, similar to the effect of JNK kinase inhibitor SP600125 after axotomy in DRG neurons (Fig. 4, B and C), which is consistent with previous findings (14).
Then we examined the effect of Spy1 on injury-induced axonal degeneration. As shown in Fig. 4D, Spy1 knockdown significantly delayed axonal degeneration as quantified by degeneration index, a measurement of fragmented axonal area calculated from phase-contrast images (27,28). In neurons infected with control virus, axonal fragmentation was apparent by 6 h and robust by 9 h after axotomy (Fig. 4E). In contrast, following knockdown of Spy1, degeneration was significantly delayed (Fig. 4E). Reintroduction of exogenous Spy1 restored axonal degradation, whereas SP600125 treatment halted axonal degradation (Fig. 4E). Collectively, these results demonstrated that depletion of Spy1 attenuated SCG10 phosphorylation and delayed injury-induced axonal degeneration.

Discussion
Axon loss is a destructive consequence of a wide range of neurological diseases. Therapies targeting the axon loss process itself are notably absent. Clarifying the mechanism of axon loss may help develop such therapies. Regulated protein degradation promotes the degeneration of injured axons (3), potentially via the degradation of vulnerable axonal maintenance factors. Recent data have demonstrated that SCG10, being similar to NMNAT2 (29), is a novel axonal maintenance factor (14); rapid SCG10 loss after injury requires JNK activity. However, pharmacologically inhibiting JNK activity further slows the degradation of the mutant SCG10 in which two JNK phosphorylation sites (serines 62 and 73) were replaced by alanines, demonstrating that JNK may promote the degradation of SCG10 through other mechanisms, in addition to the phosphorylation of SCG10 (14). Therefore, understanding the mechanisms regulating SCG10 stability may be helpful in finding ways to attenuate axonal destruction, for example, by inhibiting specific degradation machinery targeting SCG10.
Spy1, as a Speedy/RINGO family protein, is implicated in mammary development (21) and cell cycle regulation. In addition, our previous data have showed that Spy1 expression could be detected in the lumbar spinal cord, including neurons and glial cells (22), and that Spy1 may participate in the pathological process response to sciatic nerve injury (23). To identify the possible role of Spy1 in the nervous system, we performed yeast two-hybrid screening in a human fetal brain complementary DNA library and found that SCG10 was a binding partner of Spy1. As SCG10 plays an important role in axonal injury, we speculate that Spy1 affects axonal injury in a SCG10-dependent manner. Our work further showed Spy1-mediated SCG10 phosphorylation and injury-induced axonal degeneration, which confirmed our hypothesis.
Previous data showed that JNK phosphorylation of SCG10 targeted SCG10 for degradation (14), and our data showed that Spy1 increased JNK kinase activity. These data are in line with previous reports that Spy1 was an activator of CDKs (15,16), suggesting that Spy1 may exert its pathobiological functions via modulating kinase activities. On the other hand, recent data showed that dual leucine zipper kinase (DLK) regulated stressinduced JNK activity in axons via interaction of dual leucine zipper kinase with the scaffolding protein JIP3 (30,31). Whether Spy1 has an identical role like dual leucine zipper kinase needs further studies. Furthermore, treatment with JNK kinase inhibitor SP600125 only partially abolished Spy1-mediated phosphorylation of SCG10, suggesting that other mechanisms may take part in the Spy1-mediated phosphorylation of FIGURE 4. Inhibition of Spy1 attenuates SCG10 phosphorylation and delays injury-induced axonal degeneration. A, down-regulation of Spy1 in DRG neurons by lentiviral shRNA. Lysates were prepared and analyzed by Western blotting for Spy1 and the GAPDH loading control. B, DRG cultures were axotomized (cut) and treated with control lentiviral shRNA, Spy1 shRNA #3, or 15 M SP600125 for 3 h, and Spy1 expression was restored by coexpressing human Spy1 cDNA with the rat-specific Spy1 shRNA #3. The distal axons were subjected to Western blot analysis to assess the gel mobility of SCG10. Spy1 shRNA-treated axons preferentially preserve lower molecular weight SCG10, similar to the effect of JNK kinase inhibitor SP600125. ␤3-Tubulin is shown as a loading control. C, the ratio of SCG10 protein relative to ␤3-tubulin by densitometry. The data are mean Ϯ S.E. (n ϭ 3, *, #, p Ͻ 0.01, significantly different from the uncut group). D, phase-contrast images of axons were taken 12 h after axotomy from the DRG cultures infected with the indicated treatment. E, degeneration indices were obtained from the results in B and plotted against time after axotomy. At 0, 9, and 12 h after axotomy, n ϭ 12-15; 24 h after axotomy, n ϭ 6-8; *, #, p Ͻ 0.05, ***, ###, p Ͻ 0.001 versus control lentivirus by analysis of variance. Error bars represent S.E. n.s., not significant.
SCG10. Clearly, more work is warranted to address this lingering issue.
Most studies show that Spy1 is a cell cycle regulator and that it promotes tumorigenesis (15)(16)(17)(18)(19)(20)(21). Here, we demonstrate a cell cycle-independent role for Spy1. Two pieces of evidence may support our data. First, SCG10 binds to the Speedy/RINGO domain of Spy1, but not the CDK-binding domain on the N terminus. Second, the functional interplay between Spy1 and SCG10 is staged in the axons instead of the nucleus where cell cycle regulation takes place. Further investigation is needed to clarify whether SCG10 might modulate Spy1 reciprocally in the nucleus.
In conclusion, our data indicate that Spy1 is an important regulator of axonal maintenance factor SCG10. As such, understanding its regulatory mechanisms may help to find new methods for attenuating axonal degeneration. Spy1 may be a novel axo-protective therapeutic target for axon loss.