Tankyrase Regulates Neurite Outgrowth through Poly(ADP-ribosyl)ation-Dependent Activation of β-Catenin Signaling

Poly(ADP-ribosyl)ation is a post-translational modification of proteins by transferring poly(ADP-ribose) (PAR) to acceptor proteins by the action of poly(ADP-ribose) polymerase (PARP). Two tankyrase (TNKS) isoforms, TNK1 and TNK2 (TNKS1/2), are ubiquitously expressed in mammalian cells and participate in diverse cellular functions, including wnt/β-catenin signaling, telomere maintenance, glucose metabolism and mitosis regulation. For wnt/β-catenin signaling, TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, a key component of the β-catenin degradation complex, which allows Axin’s ubiquitination and subsequent degradation, thereby activating β-catenin signaling. In the present study, we focused on the functions of TNKS1/2 in neuronal development. In primary hippocampal neurons, TNKS1/2 were detected in the soma and neurites, where they co-localized with PAR signals. Treatment with XAV939, a selective TNKS1/2 inhibitor, suppressed neurite outgrowth and synapse formation. In addition, XAV939 also suppressed norepinephrine uptake in PC12 cells, a rat pheochromocytoma cell line. These effects likely resulted from the inhibition of β-catenin signaling through the stabilization of Axin, which suggests TNKS1/2 enhance Axin degradation by modifying its poly(ADP-ribosyl)ation, thereby stabilizing wnt/β-catenin signaling and, in turn, promoting neurite outgrowth and synapse formation.


Introduction
Poly(ADP-ribose) polymerase (PARP) catalyzes ADP-ribosylation, a reversible posttranslational protein modification in which one or more ADP-ribose units are transferred from donor NAD + to target proteins [1][2][3]. This modification alters the activity and subcellular localization of the target proteins, thereby regulating diverse cellular functions. The PARP family consists of 17 members that differ in their localization and enzymatic activity. Among them, tankyrase 1 and 2 (TNKS1/2), also known as PARP5a and PARP5b, respectively, each contain two unique structural domains not contained by other PARP members: an ankyrin repeat domain and a sterile alpha motif (SAM). The ankyrin repeat domain serves as the interface for interaction with acceptor proteins, while the SAM regulates the polymerization of TNKS [4][5][6][7]. As TNKS1 and 2 are highly homologous and exhibit similar localizations at telomers, centrosomes, nuclear pores, Golgi complexes, cytoplasm and peroxisomes, their functions appear to overlap. Consistent with that idea, deletion of the gene encoding either TNKS1 or TNKS2 does not result in significant changes in mice, but their double knockout is embryonically lethal [8,9]. As implied by the lethality of TNKS1/2 double deletion, TNKS1/2-catalyzed poly(ADP-ribosyl)ation is involved in a variety of essential physiological processes. For example, knockdown of TNKS1 results in mitotic arrest, suggesting that TNKS1 is required for sister chromatid resolution, which is necessary for mitotic progression [10,11]. TNKS1/2 are also known to regulate the wnt/β-catenin pathway involved in cell proliferation and differentiation [12][13][14]. The cellular level of β-catenin is controlled by the β-catenin destruction complex composed of Axin, adenomatosis polyposis coli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α) [15,16]. Two serine residues on β-catenin are phosphorylated by GSK3β, which allows its ubiquitination by TrCP1, an E3 ubiquitin ligase, and its subsequent proteasomal degradation [17,18]. TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, enabling its ubiquitination by RNF146, an E3 ubiquitin-protein ligase, and proteasomal degradation [13,19]. Axin degradation disrupts the β-catenin destruction complex, after which the stabilized β-catenin is translocated to the nucleus, where it binds to TCF/LEF, which mediates the transcription of several genes. TNKS1/2 are ubiquitously expressed in mammalian cells, especially in the brain, ganglia, skin, and heart. However, the function of TNKS1/2 in the nervous system remains unclear due in large part to the embryonic lethality of TNKS1/2 double deletion. In the present study, however, we investigated the effect of TNKS1/2 on neurite outgrowth in mouse primary hippocampal neurons and rat adrenal medullary neuron-like cells, PC12 cells.

TNKS1/2-Mediated Neurite Outgrowth and Synapse Formation in Primary Hippocampal Neurons
To understand the role of PARP isoforms in neurons, beginning one day after the start of culture, primary hippocampal neurons were incubated with various PARP inhibitors, including ABT888, a selective PARP1 and PARP2 inhibitor; PJ34, a nonselective PARP inhibitor; and XAV939, a selective TNKS1/2 inhibitor. The neurites were stained with anti-MAP2 antibody to measure their length and number. PJ34 and XAV939, but not ABT888, suppressed neurite length significantly after 5 and 7 days in vitro (DIV), though they did not affect the number of neurites sprouting from the soma ( Figure 1A,B). XAV939 also reduced the number of neurite branches significantly after 7 DIV ( Figure 1B). In addition, PJ34 and XAV939, but not ABT888, suppressed the density of presynaptic staining with anti-synaptophysin antibody and postsynaptic staining with anti-PSD95 antibody on the anti-MAP2 antibody-stained neurites ( Figure 1C,D). As XAV939 and PJ34 exert inhibitory effects on TNKS1/2, these results indicate that TNKS1/2, but not PARP1, are involved in neurite outgrowth and synapse formation, but not neurite sprouting. TNKS1/2 were present in the soma and neurites of primary hippocampal neurons after 5 DIV ( Figure 1E). The enzymes were distributed in a punctate pattern within neurites, where they co-localized with PSD-95, suggesting they localize to synapses ( Figure 1E, lower). Consistent with the observed distribution of TNKS1/2, poly(ADP-ribose) (PAR) was detected in the soma and at synapses on neurites ( Figure 1F). Treatment with XAV939 for 24 h suppressed the PAR levels seen in those regions ( Figure 1G,H), which suggests TNKS1/2 localize to the soma and neurites, where they catalyze poly(ADP-ribosyl)ation.

TNKS1/2 Participate in Neurite Outgrowth in PC12 Cells
To investigate the intracellular mechanism by which TNKS1/2 regulate neurite outgrowth in neurons, PC12 rat pheochromocytoma cells were used as a model. After 4 days of nerve growth factor (NGF) treatment in low-serum medium, neurite outgrowth was observed ( Figure 2A). As in hippocampal neurons, TNKS1/2 were localized in cell bodies and neurites (Figure 2A), and PJ34 and XAV939, but not ABT888, which suppressed MAP2-positive neurite outgrowth (Figure 2A,B). This suggests TNKS1/2 participate in neurite outgrowth in PC12 cells as they do in primary hippocampal neurons. PC12 cells synthesize and release catecholamines and then take them up [20]. To assess the effect of XAV939 on those actions, PC12 cells were incubated with [ 3 H]-labeled norepinephrine (NE), after which the [ 3 H]-NE content of the cells and its secretion were measured. Interestingly, XAV939 had no effect on Ca 2+ -dependent or -independent [ 3 H]-NE release ( Figure 2C), but markedly inhibited [ 3 H]-NE uptake ( Figure 2D). NE transporter (NET) mediates NE uptake into cells, and Western blotting and real-time PCR revealed that XAV939 did not alter levels of NET protein or mRNA ( Figure 2E-G). Apparently, XAV939 inhibits NE uptake by suppressing NET activity but not its expression.

TNKS1/2 Inhibition Upregulates Its Expression
We next examined the effects of PARP inhibitors on expression of TNKS1/2 protein. We found that XAV939 markedly increased levels of TNKS1/2, while PJ34 increased only TNKS2 and ABT888 had no effect ( Figure 3A,B). XAV939 increased levels of TNKS1/2 over time so that their highest levels were detected after 24 h, the last measurement made ( Figure 3C,D). Given that none of the PARP inhibitors affected TNKS1/2 mRNA expression ( Figure 3E), it appears that PARP inhibitors do not affect TNKS1/2 transcription. In addition, the proteasome inhibitor MG 132 also increased TNKS1/2 levels in PC12 cells ( Figure 3F,G), which suggests TNKS1/2 enzyme activity is likely to be involved in its own degradation by the proteasome.

Discussion
In this study, we found that TNKS1/2 are present on the cell bodies and neurites of primary hippocampal neurons and participate in neurite elongation and synapse formation. Using PC12 cells to investigate the mechanism by which TNKS1/2 affect neurite outgrowth, it was found that activation of wnt/β-catenin signaling through poly(ADP-ribosyl)ationmediated degradation of Axin1 is a key factor.
The PARP family has 17 members with different activities and subcellular localizations and specific biological activities associated with both normal physiological and pathophysiological processes [1][2][3]. Indeed, inhibition of PARP activity has clinical benefits in several diseases [24][25][26][27][28]. PARP inhibitors have been developed based on the unique structural features of each isoform, especially structural differences in the NAD + -binding domain [28,29]. Several of these inhibitors are currently in clinical trials or are now being used in clinical practice [25,[30][31][32]. In particular, PARP1-and PARP2-specific inhibitors are being used in combination with conventional anticancer drugs to promote synthetic lethality in cancer cells with BRCA1/2 mutations [33,34]. The PARP inhibitors used in the present study (PJ34, ABT888, and XAV939) have differing inhibition profiles against PARP members: ABT888 is 1000-fold more specific for PARP1/2 than TNKS1/2, while XAV939 is 200-fold more specific for TNKS1/2 than for PARP1 [28,29]. PJ34 is relatively nonspecific but shows slightly stronger inhibition against PARP1 and PARP2. Among these three inhibitors, XAV939 exhibits by far the greatest ability to inhibit elongation of hippocampal neurons and PC12 cells, as their relative efficacies are XAV939 > PJ34 > > > ABT-888. This suggests TNKS1/2, but not PARP1/2, are involved in neurite outgrowth, which is consistent with the earlier finding that mice lacking PARP1 grow normally and show no abnormalities in brain development [35].
It has been reported that wnt/β-catenin signaling contributes to the regulation of stem cell pluripotency and cell fate during development in addition to neuronal axon guidance and synapse formation [16,34,36]. As mentioned, β-catenin is phosphorylated by the βcatenin destruction complex consisting of APC, Axin1 and GSK3β, which is maintained at low levels through targeted ubiquitination and proteasomal degradation [4,15,16]. Once TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin1, it is recognized by the E3 ubiquitin ligase RNF146, which contains a PAR-binding WWE domain, leading to ubiquitinationdependent degradation [13,19]. In the present study, XAV939 suppressed poly(ADPribosyl)ation of Axin1 in PC12 cells. The resultant stabilization of Axin1 led to decreased levels of active unphosphorylated β-catenin and suppression of its nuclear translocation.
β-catenin also appears to regulate the expression of NrCAM in PC12 cells. The NrCAM promoter contains several binding sites for TCF/LEF, transcription factors required for optimal activation by β-catenin [21][22][23]. NrCAM is mainly expressed in the nervous system and is involved in nerve adhesion and neurite outgrowth [21][22][23]. When an anti-NrCAM antibody was injected into the central canal of the spinal cord of embryonic chicks, commissural axons in the spinal cord failed to extend along the longitudinal axis [22], suggesting that NrCAM plays a key role in axonal guidance. In addition to transcriptional regulation, β-catenin links cadherin to β catenin and cytoskeletal actin to stabilize cell adhesion [37,38]. Because TNKS1/2 are localized in both the soma and neurites, especially at synapses, their activation in neurons may regulate NrCAM transcription in the soma in addition to cadherin stabilization by β-catenin in neurites, both of which are involved in neurite outgrowth and synapse formation ( Figure 6). Figure 6. Tankyrase activates β-catenin signaling to enhance neurite outgrowth and synapse formation. TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin and TNKS1/2 themselves, which activates β catenin signaling. Once translocated to the nucleus, β-catenin promotes transcription of NrCAM gene. β-catenin is also involved in stabilizing cadherin on the cell membrane. TNKS1/2-mediated upregulation of adhesion molecules may enhance neurite outgrowth and synapse formation.
Inhibition of TNKS1/2 in PC12 cells also suppressed NE uptake, but not NE release. NE uptake is primarily mediated by NET [39]. The fact that XAV939 did not suppress NET expression suggests that it suppresses the subcellular localization and/or activity of NETs. In adipocytes, XAV939 impairs vesicle trafficking and translocation of glucose transporter 4 (GLUT4) to the plasma membrane, leading to suppression of insulin-induced glucose uptake [40]. This suggests that TNKS1/2 regulate vesicle transport and that they are involved in delivering to synapses the proteins necessary for synapse formation/maintenance and synaptic transmission (such as NET).

Cell Culture
Primary cultures of hippocampal neurons were prepared from neonatal (P0) C57BL/6JJ mice using the SUMITOMO Nerve-Cell Culture System (Sumitomo Bakelite, Tokyo, Japan). Hippocampal neurons were then cultured in Neurobasal Medium (Thermo Fisher Scientific) containing 2% B-27 Supplement (Thermo Fisher Scientific) at 37 • C and 5% CO 2 , replacing half the volume of the medium every 5 days.

Real-Time PCR
PC12 cells were cultured in 12-well plates (2 × 10 5 cells) at 37 • C and 5% CO 2 . Total mRNA was extracted using Sepazole RNA II Super, and cDNA was synthesized using a S1000 thermal cycler (Bio-rad, Hercules, CA, USA) with a Prime Script RT reagent kit. The PCR products were analyzed with Thermal Cycler Dice (Takara Bio) using SYBR Premix Ex Taq II and predesigned primers.

SDS-PAGE and Western Blotting
PC12 cells (5 × 10 5 cells) cultured in 6-well plates at 37 • C and 5% CO 2 were lysed in 50 mM Tris-HCl (pH 7.4) containing 2% SDS. After adjusting the protein concentration in the lysate, samples were prepared with NuPAGE LDS sample buffer (4×, Thermo Fisher Scientific) and NuPAGE sample reducing agent (10×, Thermo Fisher Scientific), electrophoresed on 4-12% NuPAGE bis-tris gel (Thermo Fisher Scientific), and transferred to nitrocellulose membranes (Thermo Fisher Scientific). The membranes were then blocked with Blocking One for 1 h at 37 • C, reacted with primary antibody overnight at 4 • C, washed with TBS containing 0.1% Tween 20, treated with secondary antibody (anti-rabbit or antimouse antibody) for 1 h at room temperature, washed again with TBS containing 0.1% Tween 20, and treated with an additional secondary antibody (anti-rabbit or anti-mouse antibody, HRP conjugate, (Promega, Madison, MI, USA)) for 1 h at room temperature. Finally, the membranes were reacted with SuperSignal West (Thermo Fisher Scientific), and chemiluminescence was detected using an Amersham Imager 600.

Statistical Analysis
Statistical analysis was performed using Sigmaplot (Systat Software Inc., Chicago, IL, USA). Significance was determined using paired t-tests or Student's t-test for pairwise comparison or one-way ANOVA with post hoc Dunnett's test. Data are presented as means ± S.E.M of values from the indicated numbers of experiments. Values of p < 0.05 were considered significant. All representative experiments were repeated three times.