PLPP/CIN-mediated NF2-serine 10 dephosphorylation regulates F-actin stability and Mdm2 degradation in an activity-dependent manner

Neurofibromin 2 (NF2, also known as merlin) is a tumor suppressor protein encoded by the neurofibromatosis type 2 gene NF2. NF2 is also an actin-binding protein that functions in an intrinsic signaling network critical for actin dynamics. Although protein kinase A (PKA)-mediated NF2-serin (S) 10 phosphorylation stabilizes filamentous actin (F-actin), the underlying mechanisms of NF2-S10 dephosphorylation and the role of NF2 in seizures have been elusive. Here, we demonstrate that pyridoxal-5′-phosphate phosphatase/chronophin (PLPP/CIN) dephosphorylated NF2-S10 site as well as cofilin-S3 site. In addition, NF2-S10 dephosphorylation reversely regulated murine double minute-2 (Mdm2) and postsynaptic density 95 (PSD95) degradations in an activity-dependent manner, which increased seizure intensity and its progression in response to kainic acid (KA). In addition, NF2 knockdown facilitated seizure intensity and its progress through F-actin instability independent of cofilin-mediated actin dynamics. Therefore, we suggest that PLPP/CIN may be a potential therapeutic target for epileptogenesis and NF2-associated diseases.


Introduction
Neurofibromin 2 [NF2, also known as merlin (moesinezrin-radixin-like protein) or schwannomin] is a tumor suppressor protein encoded by the neurofibromatosis type 2 gene NF2. Deletion or loss-of-function mutation of NF2 causes neurofibromatosis type 2, which is a dominant inherited disorder characterized by the development of multiple benign tumors in the nervous system. The most common tumors found in neurofibromatosis type 2 are schwannoma, meningioma, and ependymoma [1][2][3] . NF2 is also an actin-binding protein that links membrane proteins to cytoskeleton and functions in an intrinsic signaling network critical for actin dynamics in various cells [4][5][6] .
Filamentous actin (F-actin) plays an important role in stabilization and structural modification of dendritic spines that are critical structural and functional components of neurons receiving and integrating the majority of excitatory synaptic inputs [7][8][9][10][11] . Indeed, F-actin polymerization (by jasplakinolide) increases seizure threshold response to picrotoxin, while depolymerization (by latrunculin A) decreases it 12 . In the brain, NF2 expresses in dendrites 13 , axons 14,15 , the cytoplasm 16,17 , and synaptic junctions 18 of cortical and hippocampal neurons. Interestingly, c.428_430delCTTdel mutation in Nf2 is associated with a predisposition to development of benign brain tumors in which the on-set of symptoms is characterized by status epilepticus (SE, a prolonged seizure activity) in humans 19 . Furthermore, valproic acid (an antiepileptic drug) up-regulates NF2 expression 20 . With respect to NF2-actin interactions [4][5][6] . it is likely that NF2mediated actin dynamics would play an important role in the regulation of neuronal excitability. However, the role of NF2 in F-actin stabilization is still controversial, although NF2 is involved in actin dynamics. NF2 stabilizes F-actin and reduces its depolymerization rates 21 . In contrast, loss of function of NF2 inhibits F-actin severing and depolymerizing activity of cofilin by increasing LIM domain kinase-1 (LIMK1)-mediated serine (S) 3 phosphorylation 22,23 .
On the other hand, NF2 activity is regulated by phosphorylations. C-terminal S518 site is phosphorylated both by protein kinase A (PKA) and p21-activated kinase (PAK) 24,25 . Phosphorylation at this residue inhibits NF2 tumor suppressor activity by blocking its head-to-tail interaction 24 , which leads to cell growth and cell division 26,27 . Indeed, S518 dephosphorylation by the myosin phosphatase activates NF2 that promotes growth arrest and neurite outgrowth 13,26,28 . S518 phosphorylation also weakens the NF2-cytoskeleton associations 24 . In contrast, PKA-mediated NF2-S10 phosphorylation stabilizes actin filaments 29 . Therefore, the selective phosphorylations at S10-and S518 site play reverse roles in NF2-associated actin dynamics. However, little is known yet to explain the underlying mechanisms of NF2-S10 dephosphorylation and the role of NF2 phosphorylation in seizure susceptibility and/or epilepsy.
Here, we demonstrate that PLPP/CIN bound to NF2 and dephosphorylated its S10 site without altering PKA activity, which reduced F-actin stability. In addition, NF2-S10 dephosphorylation reversely regulated Mdm2 and PSD95 degradations in an activity-dependent manner, which increased seizure intensity and its progression in response to kainic acid (KA). NF2 knockdown facilitated KA-induced seizure activity through F-actin instability and the reductions in Mdm2 and PSD95 degradation, independent of cofilin activity. These findings indicate that PLPP/CIN may increase F-actin instability and NF2mediated Mdm2 degradation, but inhibit PSD95 elimination, by dephosphorylating NF2-S10 and Mdm2-S166 site, which lead to neuronal hyperexcitability. Therefore, we suggest that PLPP/CIN may be a potential therapeutic target for epileptogenesis and NF2-associated diseases.
Since PKA simultaneously phosphorylates NF2 at S10 and S518 sites 29,40 , we could not directly investigate the effect of the modulation of PKA activity on NF2-S10 phosphorylation. However, PLPP/CIN over-expression and its deletion selectively affected NF2-S10 phosphorylation under physiological-and post-KA conditions. Thus, we analyzed the correlation between F-actin content and NF2 expression/phosphorylation levels within control siRNA-and NF2 siRNA-treated groups of WT, PLPP/CIN Tg and PLPP/CIN −/− mice under physiological conditions, instead of the direct manipulation of PKA activity. Linear regression analysis showed a direct proportional relationship between NF2 expression and Factin contents with linear correlation coefficients of 0.4237 (t (54) = 3.44, p = 0.001; Fig. 5a). The F-actin contents also showed a direct proportional relationship with NF2-S10 phosphorylation level (linear correlation coefficients, 0.7977; t (54) = 9.72, p < 0.001; Fig. 5a). However, the NF2-S518 phosphorylation showed no proportional relationship with F-actin contents (linear correlation coefficients, 0.1975; t (54) = 1.48, p = 0.1446; Fig. 5a). These findings indicate that S10 phosphorylation may reinforce NF2-mediated F-actin stability independent of cofilin activity.

Discussion
PLPP/CIN is a PLP phosphatase and a serine protein phosphatase, which activates cofilin-mediated F-actin depolymerization 30,31 . Recently, we have reported that PLPP/CIN dephosphorylates NEDD4-2, Mdm2, and calsenilin (CSEN, also known as downstream regulatory element antagonist modulator or potassium channel interacting protein 3), independent of cofilin-mediated Factin depolymerization 34,35,38 . In the present study, we found that PLPP/CIN also dephosphorylated NF2 at S10 site under physiological-and post-KA conditions. Furthermore, the NF2 protein level and NF2-S10 phosphorylation level showed a direct proportional relationship with F-actin contents. NF2 is an actin-binding protein with anti-proliferative activity that is regulated by phosphorylations. A closed clamp conformation of NF2 via intramolecular interactions of its N-terminal domain with an α-helical C-terminal domain acts as an active growth suppressor. The phosphorylation of serine 518 by PKA and/or PAK1 leads to an open conformation and inhibits anti-proliferative and actin-binding activities of NF2 24 . In contrast, the first 18 amino acids of NF2 are involved in actin-binding 42 , and PKA-mediated S10 phosphorylation enhances NF2-actin binding and F-actin stability 29 . Given the role of NF2-S10 phosphorylation in F-actin stability 29 , our findings suggest that PLPP/CIN may reduce F-actin stabilization and filament assembly by dephosphorylating NF2 and cofilin at S10 and S3 site, respectively.
While NF2 selectively binds and stabilizes actin filaments 21,43 , loss of NF2 function paradoxically abrogates Factin disassembly by increasing LIMK1-mediated cofilin inactivation 22,23 . In the present study, NF2 knockdown reduced F-actin contents in PLPP/CIN Tg and PLPP/CIN −/− mice, although it did not affect p-S10 NF2 ratio and cofilin phosphorylation in both groups. In addition, the NF2 protein level and the p-S10 NF2 ratio showed a direct proportional relationship with F-actin contents. Since PLPP/ CIN over-expression and its deletion do not change LIMK1 activity 11 , these findings suggest that NF2 itself may interfere with cofilin-actin bindings and/or reduce the yield of cofilin-mediated F-actin depolymerization, independent of LIMK1 activity.
F-actin is one of the most abundant cytoskeletal proteins in dendritic spines, which regulates spine morphogenesis and synaptic strength 44,45 . Indeed, F-actin depolymerization in dendritic spines increases neuronal excitability and decreases seizure thresholds 12,32 . In our previous study 34 , PLPP/CIN Tg mice show the more severe seizure intensity and the prolonged seizure progression in response to KA, while PLPP/CIN Tg mice interrupt these phenomena. Furthermore, jasplakinolide (an F-actin stabilizer) ameliorates seizure intensity (total EEG power) in PLPP/CIN Tg mice, while latrunculin A (an F-actin depolymerizer) aggravates it in PLPP/CIN −/− animals. Thus, we have reported that PLPP/CIN-mediated F-actin depolymerization contributes to the seizure intensity and its progression in response to KA. Similar to latrunculin A treatment, the present study reveals that NF2 knockdown increased seizure intensity in response to KA in WT, PLPP/CIN Tg , and PLPP/CIN −/− mice, and its efficacies on seizure activity were PLPP/CIN Tg > WT > PLPP/CIN −/− mice. Considering the effects of NF2 knockdown on its phosphorylation ratios, F-actin contents, and cofilin activity, these findings indicate that NF2 itself and/or NF2-S10 phosphorylation may attenuate seizure activity in response to KA through F-actin stability, independent of cofilin activity. Therefore, our findings suggest that PLPP/CIN-mediated NF2 dephosphorylation may serve as one of the mechanisms by which F-actin instability mediates neuronal hyperexcitability.
the roles of p53 in seizure activity have been still controversial [48][49][50][51][52] . The stabilization of p53 by NF2 is accomplished through Mdm2 degradation and the Nterminal region of NF2 is responsible for this activity 39,53 . Therefore, we hypothesized that NF2 would attenuate KA-induced seizure activity by regulating Mdm2-p53 signaling pathway, although little is currently known regarding the contribution of NF2-Mdm2 axis to the neuronal excitability. In the present study, NF2 knockdown attenuated KA-induced Mdm2 degradation in WT and PLPP/CIN Tg mice, which was abrogated by PLPP/CIN deletion. Aforementioned, however, the present data show that NF2 siRNA exacerbated KA-induced seizure activity, similar to the cases of Mdm2 knockdown 38 . Since both PLPP/CIN and Mdm2 do not affect protein level of p53 in post-mitotic neurons under physiological-and post-KA conditions 38 , it is likely that NF2 may regulate seizure activity independent of the Mdm2-p53 signaling pathway.
Consistent with a previous study 38 , the present study demonstrates that PLPP/CIN dephosphorylated Mdm2-S166 site, and facilitated its degradation induced by KA injection. Furthermore, KA increased NF-S10 dephosphorylation, which was exerted by PLPP/CIN overexpression. Interestingly, NF2 also decreases Mdm2-S166 phosphorylation by inhibiting AKT activity 54 . Thus, it is plausible that NF2 itself and/or NF2-S10 phosphorylation would directly affect AKT-mediated Mdm2-S166 phosphorylation. However, the present data show that NF2 siRNA did not change Mdm2-S166 phosphorylation under physiological condition. Furthermore, PLPP/CIN over-expression and its deletion do not affect AKT activity under physiological-and post-KA conditions 38 . Therefore, it is excluded the possibility that the NF2mediated AKT regulation would affect Mdm2-S166 phosphorylation in PLPP/CIN Tg and PLPP/CIN −/− mice.
In the present study, NF2 knockdown did not affect Mdm2-mediated PSD95 degradation induced by KA, although it increased the Mdm2 protein level. Considering no effect of NF2 knockdown on Mdm2-S166 phosphorylation, these findings indicate that Mdm2-S166 phosphorylation may be required for Mdm2-mediated PSD95 degradation. The present study also demonstrates that following KA injection NF2 protein level and NF2-S10 phosphorylation showed an inverse and a direct proportional relationship with the Mdm2 protein level in the presence of PLPP/CIN, respectively. Furthermore, the NF2-S10 phosphorylation level, but not the NF2 protein and S518 phosphorylation levels, showed an inverse proportional relationship with the PSD95 protein level. Regardless of the roles of AKT, p53 and NEDD4-2 in seizure activity, our findings suggest that PLPP/CINmediated NF2-S10 dephosphorylation may increase KAinduced seizure activity by enhancing F-actin instability and Mdm2-mediated PSD95 degradation (Fig. 8c).
F-actin acts as an anchor for PSD scaffolding proteins 61,62 . Furthermore, F-actin forms obstacles and barriers for the flux of synaptic molecules, which influence synaptic activity 63 . Indeed, F-actin lattice hinders the redistribution and translocations of postsynaptic proteins, and the receptor-bindings with PSD-related regulatory molecules including PSD95 (refs. 8,11,61 ). With respect to these reports, it is likely that PLPP/CIN may eliminate the local F-actin barrier by dephosphorylations of cofilin and NF2, which may provide a critical window of opportunity allowing translocations and interactions between NMDAR and PSD95. Indeed, PLPP/CIN Tg mice show the enhanced NMDAR functionality by increasing NMDAR-PSD95 co-assembly 11 . In addition, PLPP/CIN may inhibit Mdm2 activity as an E3 ubiquitin ligase for PSD95 by enhancing its S166 dephosphorylation and NF2-mediated Mdm2 degradation. These PLPP/CIN functions may increase NMDAR-mediated neuronal excitability, which would lead to the enhanced seizure intensity and its progression (Fig. 8c).
As previously stated, PLPP/CIN also regulates seizure progression and intensity in response to KA through Mdm2-and NEDD4-2-medaited PSD95 and AMPAR GluA1 subunit ubiquitination, respectively 35,38 . Indeed, knockdown of NEDD4-2 or Mdm2 increases KA-induced seizure intensity in PLPP/CIN −/− mice without affecting the latency of seizure on-set 35,38 . Similar to the effect of latrunculin A on seizure intensity 34 , the present study shows that NF2 knockdown enhanced KA-induced seizure intensity, but not latency of seizure on-set, and facilitated seizure progression in response to KA in PLPP/ CIN −/− mice. These findings indicate that PLPP/CINmediated dephosphorylations of NEDD4-2, Mdm2, and NF2 may play important roles in seizure progression and propagation in response to KA. However, PLPP/CINmediated CSEN dephosphorylation reduces seizure susceptibility by activating Kv4.2 channel (an A-type K + channel), and increases seizure duration and its intensity via prolonged NMDAR activation following KA 34 . These diverse roles of PLPP/CIN in KA-induced seizure activity suggest that the seizure susceptibility (initiation) and its severity (progression) may be regulated by different and complicated mechanisms. Together with these previous studies, the present data also hypothesize that PLPP/CIN may be one of the up-stream regulators for the various signaling molecules participating the ictogenesis and seizure progression.
In conclusion, the present data provide a new implication for the interaction between the PLPP/CIN and NF2: PLPP/ CIN-mediated NF2-S10 dephosphorylation may serve as one of the mechanisms by which F-actin instability induces neuronal hyperexcitability, and Mdm2 may act as the mediator of the interaction between NF2 and PSD95. To our knowledge, this is the first report concerning the possible PLPP/CIN-NF2-Mdm2-PSD95 signaling pathway, which may lead to seizure progression and increase its severity, independent of cofilin activity and p53-NEDD4-2 axis. Therefore, we provide a new paradigm for the development of therapeutic strategies for epilepsy and neurological diseases associated with deregulation of NF2 and Mdm2.

Experimental animals and chemicals
Male PLPP/CIN −/− (129/SvEv-C57BL/6J background) and PLPP/CIN Tg (C57BL/6J background) mice (8 weeks old) were used in the present study. Each background WT mice were used as control animals for PLPP/CIN −/− and PLPP/CIN Tg mice, respectively. Animals were provided with a commercial diet and water ad libitum under controlled temperature, humidity, and lighting conditions (22 ± 2°C, 55 ± 5% and a 12:12 light/dark cycle). All experimental protocols were approved by the Animal Care and Use Committee of Hallym University (# Hallym 2018-2, 26 April 2018). All reagents were obtained from Sigma-Aldrich (USA), except as noted.

Analysis of F-actin content
To analyze F-actin content, we used G-actin/F-actin in vivo assay biochem kit (#BK037, Cytoskeleton, Inc., USA), according to the manufacturer's instructions 11 . Next, western blotting was performed according to the standard procedures (see below).

Seizure induction and EEG recording
After baseline recording for at least 30 min, animals were given KA (25 mg/kg, i.p.). Control animals received an equal volume of normal saline instead of KA. EEG signals were recorded with a DAM 80 differential amplifier (0.1-1000 Hz bandpass; World Precision Instruments, USA) and the data were digitized (1000 Hz) and analyzed using LabChart Pro v7 software (AD Instruments, Australia). Latency of seizure on-set was defined as the time point showing more than 3 s and consisting of a rhythmic discharge between 4 and 10 Hz with an amplitude of at least two times higher than the baseline EEG 34,35,38 . Total EEG power was normalized by the baseline power obtained from each animal. Spectrograms were automatically calculated using a Hanning sliding window with 50% overlap by LabChart Pro v7. Diazepam (Valium; Roche, France; 10 mg/kg, i.p.) was administered 2 h after KA injection. This is because 2-h after KA injection is the suitable time point to compare the time of seizure on-set, total EEG power and the changes in biochemical profiles in PLPP/CIN Tg and PLPP/CIN −/− mice 34,35,38 . Behavioral seizure severity was also evaluated based on the seizure score as followed: (0) no change, (1) no movement, (2) increase in muscle tone at rest, (3) head bobbing/ scratching or and circling, (4) clonus/rearing/falling of forelimb, (5) repetitive behavior of 4, (6) severe tonic-clonic seizures 48 . After recording, animals were quickly decapitated, and their hippocampi were dissected out in the presence of cooled artificial cerebrospinal fluid (in mM: 124 NaCl, 5 KCl, 1.25 NaH 2 PO 4 , 26 NaHCO 3 , 10 dextrose, 1.5 MgCl 2 , and 2.5 CaCl 2 ) and stored −80°C until preparation for biochemical experiments 34,35,38 .

Co-immunoprecipitation
The hippocampal tissues were lysed in radioimmunoprecipitation assay buffer (RIPA: 50 mM Tris-HCl pH 8.0; 1% Nonidet P-40; 0.5% deoxycholate; 0.1% sodium dodecyl sulfate (SDS), Thermo Fisher Scientific, USA) containing protease inhibitor cocktail (Roche Applied Sciences, USA), phosphatase inhibitor cocktail (PhosSTOP ® , Roche Applied Science, USA), and 1 mM sodium orthovanadate. Protein concentrations were calibrated by BCA protein assay (Pierce, USA) and equal amounts of total proteins (150 μg) were incubated with NF2 or PLPP/CIN antibody (Supplementary Table 1) and protein G sepharose beads at 4°C overnight. In vitro sample were also reacted with each antibody by the same method. Beads were collected by centrifugation, eluted in 2× SDS sample buffer, and boiled at 95°C for 5 min. Thereafter, the samples were used for western blot.

Western blot
Western blotting was performed according to standard procedures. Briefly, sample proteins (10 μg) were separated on a Bis-Tris SDS-polyacrylamide electrophoresis gel (SDS-PAGE). Separated proteins then were transferred to polyvinylidene fluoride membranes. The membranes were incubated with a relatively specific primary antibody (Supplementary Table 1). The ECL Kit (GE Healthcare Korea, Seoul, South Korea) was used to detect signals. The bands were detected and quantified on ImageQuant LAS4000 system (GE Healthcare Korea, Seoul, South Korea). The rabbit anti-β-actin was used as a loading control for quantitative analysis of relative expression levels of proteins. The ratio of phosphoprotein to total protein was described as the phosphorylation ratio.

FJB staining and cell counting
One day after KA injection, animals were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under urethane anesthesia (1.5 g/kg, i.p.). Brains were post-fixed in the same fixative overnight and then cryoprotected and sectioned at 30 μm with a cryostat. Thereafter, tissues were used for a conventional Fluoro-Jade B (FJB) staining according to previous studies 34,35,38 . All images were obtained using an AxioImage M2 microscope and AxioVision Rel. 4.8 software. Areas of interest (1 × 10 5 μm 2 ) were selected in the captured images of the CA3 region of the hippocampus proper (10 sections per each animal). Two different investigators who were blind to the classification of tissues performed the cell count of FJB-positive neurons 34,35,38 .

Statistical analysis
Number (n) of each experimental group used for the evaluation was seven. The data obtained from each group were analyzed. After evaluating the values on normality using Shapiro-Wilk W test, two-tailed Student's t-test, repeated measures ANOVA, one-way ANOVA, and twoway ANOVA were used to analyze statistical significance. Bonferroni's test was applied for post hoc comparisons. A p value below 0.05 was considered statistically significant.