Nuclear Translocation of Calcium/Calmodulin-dependent Protein Kinase IIδ3 Promoted by Protein Phosphatase-1 Enhances Brain-derived Neurotrophic Factor Expression in Dopaminergic Neurons*

Background: The physiological function of CaMKIIδ3 phosphorylation at Ser332 remains unclear. Results: CaMKIIδ3 dephosphorylation at Ser332 promotes its nuclear localization and stimulates BDNF expression. Conclusion: Dopamine D2 receptor stimulation triggers CaMKIIδ3 dephosphorylation at Ser332 by PP1. Significance: CaMKIIδ3 (Ser332) dephosphorylation is critical for neurite extension and survival of dopaminergic neurons. We have reported previously that dopamine D2 receptor stimulation activates calcium/calmodulin-dependent protein kinase II (CaMKII) δ3, a CaMKII nuclear isoform, increasing BDNF gene expression. However, the mechanisms underlying that activity remained unclear. Here we report that CaMKIIδ3 is dephosphorylated at Ser332 by protein phosphatase 1 (PP1), promoting CaMKIIδ3 nuclear translocation. Neuro-2a cells transfected with CaMKIIδ3 showed cytoplasmic and nuclear staining, but the staining was predominantly nuclear when CaMKIIδ3 was coexpressed with PP1. Indeed, PP1 and CaMKIIδ3 coexpression significantly increased nuclear CaMKII activity and enhanced BDNF expression. In support of this idea, chronic administration of the dopamine D2 receptor partial agonist aripiprazole increased PP1 activity and promoted nuclear CaMKIIδ3 translocation and BDNF expression in the rat brain substantia nigra. Moreover, aripiprazole treatment enhanced neurite extension and inhibited cell death in cultured dopaminergic neurons, effects blocked by PP1γ knockdown. Taken together, nuclear translocation of CaMKIIδ3 following dephosphorylation at Ser332 by PP1 likely accounts for BDNF expression and subsequent neurite extension and survival of dopaminergic neurons.

In the rat brain, CaMKII␣B and CaMKII␦3 are expressed in nuclei of neurons (6,10). The activity of both is reportedly regulated by the NLS motif, which, when phosphorylated, prevents nuclear localization. For example, the CaMKII␦3 Ser 332 residue immediately C-terminal to the NLS ( 328 KKRKS 332 ) can be phosphorylated by the CaMK family members CaMKI or CaMKIV, blocking association of CaMKII with the NLS receptor m-pendulin, therefore prohibiting nuclear localization (11).
In this study, we show that the CaMKII␦3 Ser 332 site is directly dephosphorylated by protein phosphatase 1 (PP1), promoting CaMKII␦3 nuclear translocation, and that aripiprazole (APZ), a dopamine D 2 R partial agonist, promotes CaMKII␦3 nuclear translocation and enhances BDNF expression. Overall, our findings demonstrate a critical function for CaMKII␦3 nuclear translocation in promoting survival and enhancing neurite extension of dopaminergic neurons.
Cell Culture and Transfection-Neuro-2a cells were grown in DMEM supplemented with 10% heat-inactivated FBS and penicillin/streptomycin (100 units/100 g/ml) in a 5% CO 2 incubator at 37°C. Neuro-2a cells were transfected with expression vectors using Lipofectamine 2000 (Invitrogen), and experiments were performed 48 h later as described previously (29). Primary cultures of mesencephalic neurons were established using methods described previously with slight modifications (30). Briefly, SN tissue was dissected from embryonic day 18 Wistar rats and dissociated by trypsin treatment and trituration through a Pasteur pipette. Neurons were plated on coverslips coated with poly-L-lysine in minimum essential medium (Invit-rogen) supplemented with 10% FBS, 0.6% glucose (Wako, Osaka, Japan), and 1 mM pyruvate (Sigma-Aldrich). After cell attachment, coverslips were transferred to dishes containing a glial cell monolayer and maintained in Neurobasal medium (Invitrogen) containing 2% B27 supplement (Invitrogen) and 1% GlutaMax (Invitrogen). 5 M cytosine ␤-D-arabinofuranoside (Sigma-Aldrich) was added to cultures at DIV3 (3 days in vitro) after plating to inhibit glial proliferation. Primary mesencephalic neurons were transfected with expression vectors and siRNAs using electroporation (NEPA21, Nepagene Co. Ltd., Chiba, Japan) at DIV0.
Chemically Induced Long-term Potentiation or APZ Stimulation of Primary Mesencephalic Neurons-Chemically induced long-term potentiation (c-LTP) was induced as described previously (31). Briefly, neuronal cultures at DIV10 were transferred from Neurobasal medium to extracellular solution containing 140 mM NaCl, 1.3 mM CaCl 2 , 5 mM KCl, 25 mM HEPES (pH 7.4), 33 mM glucose, 0.5 M tetrodotoxin, 1 M strychnine, and 20 M bicuculline methiodide. After 10 min in extracellular solution, cells were treated with 200 M glycine in extracellular solution for 3 min and then incubated in extracellular solution without glycine for the indicated amounts of time. For APZ stimulation, cells were incubated in Krebs-Ringer-HEPES solution containing 128 mM NaCl, 5 mM KCl, 1 mM MgSO 4 , 10 mM glucose, 2.7 mM CaCl 2 , and 20 mM HEPES for 30 min and then stimulated with 10 M APZ for the indicated times. Some cells were pretreated with 0.5 M okadaic acid (Calbiochem, San Diego, CA) for 30 min before c-LTP or APZ treatment.
In Vivo APZ Treatment-Adult 8-week-old male Wistar rats (180 -220 g) were housed under climate-controlled conditions with a 12-h light/dark cycle and provided standard food and water ad libitum. Experiments were approved by the Institutional Animal Care and Use Committee at Tohoku University. APZ (Wako, 0.3 mg/kg dissolved in 0.5% carboxymethylcellulose) or vehicle was administered to rats intraperitoneal daily for 7 days. The animals were then sacrificed, and the brain was removed and perfused with ice-cold buffer for 3 min (0.32 M sucrose, 20 mM Tris-HCl (pH 7.4)). The SN region was then dissected and subjected to immunoblotting.
Cell Fractionation-Fractionation of Neuro-2a cells was performed using the subcellular protein fractionation kit for cultured cells (Pierce, Thermo Fisher Scientific Inc.) according to the protocol of the manufacturer. The kit allows separation of cytoplasmic, membrane, nuclear soluble, and chromatinbound protein extracts. 2 ϫ 10 6 cells were washed with PBS and collected by incubation with trypsin-EDTA at 37°C for 3 min. Extraction buffer for cytoplasmic isolation containing protease inhibitors was added to cell pellets. Cells were incubated at 4°C for 10 min. Then the homogenates were centrifuged (500 ϫ g) for 5 min. The supernatants (cytoplasmic extracts) were transferred to new tubes. Membrane extraction buffer containing protease inhibitors was added to precipitants, followed by vortexing and incubation at 4°C for 10 min. The homogenates were centrifuged (3000 ϫ g) for 5 min, and the supernatants (membrane extracts) were transferred to new tubes. Nuclear extraction buffer containing protease inhibitors was added to cell pellets, followed by vortexing and incubation at 4°C for 30 min. The homogenates were centrifuged (5000 ϫ g) for 5 min, and supernatants (soluble nuclear extracts) were transferred to new tubes. Nuclear extraction buffer containing protease inhibitors, 5 mM CaCl 2 , and micrococcal nuclease (300 units) was added to cell pellets, followed by vortexing and incubation at room temperature for 15 min. The homogenates were centrifuged (15,000 ϫ g) for 5 min, and the supernatants (chromatin-bound nuclear extracts) were transferred to new tubes. Protein concentrations were estimated by Bradford assay. Rat SN tissues were fractionated into cytosolic and nuclear extracts as follows. SN samples were extracted with ice-cold low-salt buffer containing 0.15 M NaCl, 50 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 4 mM EDTA, 4 mM EGTA, 1 mM Na 3 VO 4 , 50 mM NaF, 1 mM DTT, and protease inhibitors (trypsin inhibitor, pepstatin A, and leupeptin) and centrifuged at 20,000 ϫ g for 10 min. Supernatants (the cytosol fractions) were transferred to a fresh tube, whereas pelleted crude nuclei were resuspended in ice-cold high-salt buffer containing 0.5 M NaCl, 50 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 4 mM EDTA, 4 mM EGTA, 1 mM Na 3 VO 4 , 50 mM NaF, 1 mM DTT, and protease inhibitors. After centrifugation of the latter at 20,000 ϫ g for 10 min, the supernatant was transferred to a fresh tube (nuclear fraction).
Immunoprecipitation and Immunoblotting-Immunoprecipitation and immunoblotting were performed as described previously (29). Antibodies included rabbit polyclonal antibodies against pCaMKII (Ser 332 ) (1:1000), pCaMKII (Thr 286 / Thr 287 , 1:5000) (27) CaMKII and PP1 Activity Assays-A Ca 2ϩ /CaM-dependent CaMKII activity assay was performed as described previously (34). PP1 activity was assessed using methods described previously with slight modifications (33). Briefly, frozen SN samples were homogenized using a handheld homogenizer in 200 l of homogenizing buffer containing 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4), 0.5 M NaCl, 4 mM EDTA, 4 mM EGTA, 1 mM Na 3 VO 4 , 50 mM NaF, 1 mM DTT, 2 g/ml pepstatin A, and 1 g/ml leupeptin. Insoluble material was removed by centrifugation at 20,000 ϫ g for 5 min. Calyculin A-or okadaic acidsensitive protein phosphatase activities were measured using [ 32 P]casein as substrate. The phosphatase assay was carried out in 45 l of buffer containing Tris-HCl (40 mM (pH 7.5)), BSA (1 mg/ml), EDTA (1 mM), and 1 g supernatant from SN slices in the presence of okadaic acid (1 nM) to inhibit PP2A activity or in the presence of calyculin A (100 nM) to inhibit PP1/PP2A. The reaction was initiated by adding [ 32 P]casein (1 g). After 15-min incubation at 30°C, the reaction was terminated by adding 30 l of 40% trichloroacetic acid plus 20 l of 25 mg/ml BSA. After vortexing, the mixture was kept on ice for 10 min and then centrifuged at 20,000 ϫ g for 10 min. An aliquot (20 l) of supernatant was counted for 32 P radioactivity released during the incubation. PP1 activity was determined by sub-tracting activity in the presence of calyculin A from activity in the presence of okadaic acid.
Camui-FRET Analysis-Neuro-2a cells were grown on 0.01% poly-L-lysine (Sigma-Aldrich)-coated glass bottom dishes. To monitor CaMKII activation, cells were transfected with the Camui␣ or Camui␦3 plasmids. Two days later, cells were exposed to externally applied 60 mM high-KCl in Krebs-Ringer-HEPES buffer and imaged. The wavelengths used for FRET imaging were 438/24 nm (excitation), 483/32 nm (cyan fluorescent protein emission), and 542/27 nm (YFP emission) separated by a 458-nm dichroic mirror, and analysis was performed every 3 s. Ratio values were calculated by averaging fluorescence intensity from the entire cytosolic or nuclear area. FRET images were monitored using an inverted microscope (Leica DM IRB, Japan) equipped with a charge-coupled device camera (ORCA-ER, Hamamatsu, Japan). Captured images were analyzed using the Metafluor imaging system (Molecular Devices, Sunnyvale, CA).
Quantification of Neurite Sprouting and Cell Survival in Primary Dopaminergic Neurons-Neurite sprouting was quantified as described previously (36). Briefly, primary dopaminergic neurons were stained with anti-TH antibody at DIV10, and immunofluorescence images were analyzed using a confocal laser-scanning microscope. A neurite was defined as a process arising from the soma and neurite length as the distance from the soma to the tip of the longest branch. For APZ treatment, DIV8 neurons were treated with 10 M APZ for 48 h. To assess survival, at DIV10, 1-methyl-4-phenylpyridinium (MPP ϩ , Sigma-Aldrich) was added at a final concentration of 500 M for 24 h. Surviving cells were determined by the appearance of TH and DAPI staining. Six fields (10 cells/field) under each condition were chosen randomly and photographed.
Statistical Analysis-All values are expressed as mean Ϯ S.E. Comparison between two experimental groups was made using unpaired Student's t test. Statistical significance for differences among groups was tested by one-way analysis of variety with post hoc Tukey tests. p Ͻ 0.05 was considered significant.

Results
CaMKII Ser 332 Is Dephosphorylated by PP1 in Vitro-Isoforms of the CaMKII ␣, ␤, ␥, and ␦ subunits can be distinguished in part by an 11-amino acid KRKSSSSVQMM sequence in the variable region between the regulatory and association domains. CaMKII␣B and CaMKII␦3 display this motif, and phosphorylation of CaMKII␣B and CaMKII␦3 Ser 332 blocks nuclear localization (11) (Fig. 1A). To investigate the function of CaMKII phosphorylation, we first tested the specificity of an antibody against phospho-CaMKII (Ser 332 ). To do so, we performed an in vitro phosphorylation assay using purified rat brain Ca⌴⌲⌱⌱. Conventional CaMKII␣/␤ antibodies recognized 50-and 60-kDa immunoreactive bands corresponding to the ␣ and ␤ subtypes, respectively (Fig. 1B, lane 1). Likewise, a conventional CaMKII␦ antibody detected a 57-kDa immunoreactive band corresponding to the ␦ isoform (Fig. 1B,  lane 2). CaMKII phosphorylation at ␣-Thr 286 and ␤-, ␥-, and ␦-Thr 287 corresponds to the activated form of the protein (37). A pCaMKII (Thr 286 /Thr 287 ) antibody recognized three bands with molecular masses corresponding to the ␣, ␤, and ␦ subtypes (Fig. 1B, top panel, lane 5), and these bands disappeared upon treatment with PP1, a major Ser/Thr phosphatase expressed in eukaryotic cells (Fig. 1B, top panel, lane 6). The pCaMKII (Ser 332 ) antibody recognized two immunoreactive bands, CaMKII␣B and CaMKII␦3 (Fig. 1B, bottom panel). Importantly, the Ser 332 antibody did not recognize CaMKII␤, which lacks the Ser 332 site. The two bands detected by pCaM-KII (Ser 332 ) antibody were detected in the presence of EGTA (Fig. 1B, bottom panel, lane 4), but staining intensity was increased markedly in the presence of Ca 2ϩ /CaM, which stimulates autophosphorylation (Fig. 1B, bottom panel, lane 5). PP1 treatment decreased Ser(P) 332 immunoreactivity of these bands to basal levels (Fig. 1B, bottom panel, lane 6). Detection of a basal level of phosphorylation in purified CaMKII supports the idea that CaMKII (Ser 332 ) phosphorylation is in part resistant to PP1 dephosphorylation. The pCaMKII (Ser 332 ) antibody specifically recognized phosphorylated Ser 332 because preabsorption of the antibody with a 100-fold (100 g/ml) excess amount of phosphopeptide antigen totally eliminated the immunoreactivity on the blots (Fig. 1C). Taken together, we conclude that the Ser(P) 332 antibody specifically detects CaMKII autophosphorylation at Ser 332 of CaMKII␣B and CaMKII␦3, a site dephosphorylated in part by PP1 in vitro.
PP1␣ and PP1␥1 Predominantly Regulate CaMKII␦3 Nuclear Translocation-PP1 forms a heterodimer comprised of a catalytic (PP1c) and a regulatory subunit. PP1c can form a complex with over 50 regulatory or scaffolding proteins that regulate substrate specificity and PP1c subcellular distribution (38). PP1c itself is found as four isoforms (␣, ␤, or ␦ and ␥1 and ␥2) in mammalian cells (39 -43) and three (PP1␣, PP1␤, and PP1␥1) highly expressed in the brain (44). All isoforms show nearly 90% amino acid homology and are most divergent at the N and C termini. To determine subcellular localization of these proteins in neurons, we employed confocal microscopy of the enhanced GFP (eGFP)-tagged PP1 isoforms ␣, ␤, and ␥1 in Neuro-2a cells. PP1␣-eGFP and eGFP-PP1␤ were primarily cytoplasmic but showed low levels of nuclear fluorescence. However, PP1␥1-eGFP signals were diffuse in the cytoplasm and nucleoplasm and accumulated in unidentified nuclear bodies ( Fig. 2A). Nuclear inhibitor of PP1 (NIPP1) is a ubiquitously expressed protein that blocks PP1 activity (45). Coexpression of mCherry-tagged NIPP1 with different PP1 isoforms in Neuro-2a cells had no effect on PP1 isoform localization (Fig.  2B). In agreement, treatment of cells with okadaic acid, an   AUGUST 28, 2015 • VOLUME 290 • NUMBER 35

PP1 Promotes CaMKII␦3 Nuclear Localization
inhibitor of PP1 and PP2A, also had no effect on PP1 isoform localization (data not shown), suggesting overall that, in Neuro-2a cells, PP1 localization is not altered by changes in its activity.
To further investigate differences in PP1 subcellular localization, we fractionated lysates of Neuro-2a cells transfected with eGFP-tagged PP1 isoforms into cytoplasmic, membrane, nuclear soluble, and chromatin-bound fractions. We confirmed the quality of these fractions with antibodies against calcineurin (CaN, a cytosolic protein marker), CREB (a nuclear protein marker), and MeCP2 (a chromatin-bound protein marker). High levels of both PP1␣-eGFP and eGFP-PP1␤ were seen in the cytoplasmic fraction and relatively low in other fractions. However, PP1␥1-eGFP was mainly expressed in nuclei and in chromatin fractions (Fig. 2C). We next determined whether these PP1 isoforms could alter CaMKII␦3 localization. To do so, we cotransfected Neuro-2a cells with FLAG-tagged CaMKII␦3 plus PP1 isoforms and determined CaMKII␦3 localization by immunoblotting with an anti-FLAG antibody. In cells expressing CaMKII␦3 alone, CaMKII␦3 was primarily cytoplasmic, with relatively low levels in membrane and nuclear fractions. However, in cotransfected with PP1␣ or PP1␥1, nuclear CaMKII␦3 levels increased significantly, particularly in the presence of PP1␥1. We observed no effect on CaMKII␦3 localization when PP1␤ was cotransfected (Fig. 2, D and E; n ϭ 3 each).
To monitor CaMKII activity in cells, we assessed dynamic real-time CaMKII activation using Camui, a FRET based-biosensor molecule including full-length CaMKII (28). In Camui␣transfected Neuro-2a cells, the FRET signal, an indicator of CaMKII␣ activation, was increased significantly in cytoplasmic areas but low in nuclear areas in response to high KCl stimulation (Fig. 3C). On the other hand, in Camui␦3, the FRET signal in nuclei following KCl stimulation increased markedly compared with Camui␣ (p Ͻ 0.001, n ϭ 6 -8), (Fig. 3, D and F). A similar analysis in Camui␦3 cells cotransfected with PP1␥1 demonstrated significantly increased FRET signals in nuclei compared with Camui␦3 alone (p Ͻ 0.001, n ϭ 8 -10; Fig. 3, E and F). Taken together, these analyses indicate that nuclear CaMKII␦3 activation is enhanced by PP1-dependent Ser 332 dephosphorylation.

Treatment of Cultured Dopaminergic Neurons with the D 2 R Agonist APZ Promotes CaMKII␦3 (Ser 332 ) Dephosphorylation-
We have shown previously that CaMKII␦3 is highly expressed in rat SN dopaminergic neurons (24) and that dopamine D 2 R stimulation of NG108-15 cells activates CaMKII␦3 with concomitantly increased BDNF gene expression (25). Here we used primary cultured (DIV10) mesencephalic dopaminergic neurons to find out whether the CaMKII␦ phosphorylation status changed following c-LTP or APZ treatment. Because we found that Ser 332 was increased markedly in the presence of Ca 2ϩ / CaM in vitro, we confirmed in situ whether the phosphorylation occurred on cultured mesencephalic neurons under depolarization conditions such as c-LTP. Following c-LTP, levels of phosphorylated CaMKII␦ (Thr 287 ) and CaMKII␦ (Ser 332 ) increased significantly, lasting until 60 min after stimulation (Fig. 4, A and B). APZ treatment, on the other hand, significantly decreased CaMKII␦ (Ser 332 ) phosphorylation without altering CaMKII␦ (Thr 287 ) phosphorylation (Fig. 4, A and C). Treatment of cultured neurons with the phosphatase inhibitor okadaic acid completely blocked CaMKII␦ (Ser 332 ) dephosphorylation following APZ treatment. These findings suggest that PP1/PP2A activation by APZ underlies CaMKII␦ (Ser 332 ) dephosphorylation.
APZ Treatment Enhances CaMKII␦ Nuclear Translocation in the Rat Substantia Nigra in Vivo-Next we asked whether in vivo activation of dopamine D 2 R with APZ altered CaMKII␦ localization or phosphorylation status. Consistent with our previous study (24), CaMKII␦ was expressed in both the cytosol and nuclei of rat SN dopaminergic neurons (Fig. 5A). Immunoblot analysis demonstrated significantly decreased CaMKII␦ (Ser 332 ) phosphorylation following chronic APZ treatment compared with vehicle-treated animals (p ϭ 0.017, n ϭ 4). APZ treatment, however, did not alter the levels of phosphorylated CaMKII␦ (Thr 287 ) (Fig. 5B). Next we asked whether PP1 activity could account for changes in CaMKII (Ser 332 ) phosphorylation following APZ treatment. PP1 activity was measured using [ 32 P]casein as substrate by subtracting activity in the presence of 100 nM calyculin A to inhibit PP1 and PP2A from activity in the presence of 1 nM okadaic acid to inhibit PP2A, as described previously (33). Interestingly, we observed increased PP1 activity in SN lysates from APZ-treated rats (p ϭ 0.04, n ϭ 4; Fig.  5C). Immunoblot analysis showing an increased ratio of nuclear to cytoplasmic CaMKII␦ immunoreactivity after APZ treatment confirmed CaMKII␦ nuclear translocation (p ϭ 0.04, n ϭ 3; Fig. 5D). Moreover, BDNF protein levels also increased significantly following APZ treatment (p ϭ 0.01, n ϭ 4; Fig. 5E).
Finally, we addressed whether nuclear CaMKII␦3 activation stimulates neurite extension or survival of cultured primary mesencephalic neurons at DIV10. Morphological analysis of dopaminergic (TH-positive) cells cotransfected with CaMKII␦3 and PP1␥1 indicated significantly enhanced neurite extension compared with cells transfected with WT CaMKII␦3 alone. Similarly, CaMKII␦3 (S332A) overexpression significantly stimulated neurite extension. We also assessed whether PP1␥ knockdown altered neurite extension stimulated by nuclear CaMKII␦3 in these cells. Therefore, we first tested oligomeric siRNA targeting PP1␥ (siPP1␥) and found that knockdown decreased endogenous PP1␥ expression to ϳ30% of the levels seen in cultures of mesencephalic neurons (Fig. 6C). Cells transfected with WT CaMKII␦3 plus siPP1␥ showed significantly decreased neurite extension relative to WT CaMKII␦3transfected cells not treated with siPP1␥. Importantly, following APZ treatment, neurite extension increased significantly in TH-positive neurons, and PP1␥ knockdown blocked this effect  (Fig. 6, D and E).

Discussion
Our study documents a novel mechanism underlying the nuclear translocation of CaMKII␦3 through PP1-dependent dephosphorylation following dopamine D 2 R activation. Furthermore, we report that increased nuclear CaMKII␦3 activity mediates BDNF expression, which, in turn, may promote neurite extension and neuroprotection in dopaminergic neurons. Our working model is shown in Fig. 7. Stimulation with the dopamine D 2 R agonist APZ increases PP1 activity through inactivation of the cAMP/PKA/inhibitor 1 (I-1) pathway (47), and, in turn, PP1 dephosphorylates CaMKII␦3(Ser 332 ) in the cytoplasm, enabling CaMKII␦3 nuclear translocation. On the basis of the idea that, under basal conditions, CaMKII is autonomously active in part because of spontaneous neuronal activity (48), CaMKII␦3 is autophosphorylated in the cytoplasm, and D 2 R-mediated PP1 activation mediates its dephosphorylation at Ser 332 . Thereafter, nuclear CaMKII␦3 phosphorylates transcription factors, including MeCP2 and CCAAT/enhancerbinding protein, increasing BDNF expression. Depolarization causes Ca 2ϩ entry through NMDA receptors or voltage-dependent calcium channels and promotes CaMKII␦3 autophosphorylation at Thr 287 and Ser 332 in the cytosol. Conversely, nuclear CaMKI or CaMKIV activity may account for CaMKII␦3 nuclear export through phosphorylation at Ser 332 , as reported previously (11).
In an in vitro phosphorylation assay using purified rat brain CaMKII, CaMKII␦ was dephosphorylated by PP1 at both Ser 332 and Thr 287 . However, in experiments using primary cultured mesencephalic dopamine neurons and in chronic APZ-treated rats, CaMKII␦ was dephosphorylated only at Ser 332 by activated PP1. This discrepancy may be explained by the binding of various proteins in the CaMKII-PP1 complex. For example, spinophilin targets PP1 to postsynaptic density sites (49). CaMKII autophosphorylation at Thr 286 /Thr 287 stabilizes CaMKII localization at the postsynaptic density site (50), and the postsynaptic density-associated CaMKII holoenzyme is resistant to PP1 dephosphorylation (51). Indeed, the in vitro experimental conditions used here resemble the cytosolic microenvironment, in which PP1 directly dephosphorylates cytosolic CaMKII␦3. We did not define proteins binding to the CaMKII␦3-PP1 complex in vivo. Further studies are required to precisely identify proteins that directly regulate the CaMKII␦3 phosphorylation status in the nucleus and cytoplasm.
In FRET-based CaMKII activity assays in Neuro-2a cells using the cytoplasmic isoform CaMKII␣ probe, the FRET signal in Camui␣-transfected cells was increased slightly in the nuclear region in response to stimulation with high KCl. Purified Camui␣ with a molecular mass of 110 kDa was eluted as a single peak of a molecular mass of Ͼ1000 kDa by gel filtration (28), indicating that Camui␣ expressed in Neuro-2a cells oligomerizes with other endogenous isoforms within cells. The CaMKII holoenzyme is normally a dodecameric complex formed via isoform variable regions, and its composition affects CaMKII localization (52,53). The ability of CaMKII to translocate to the nucleus is then limited by the presence of nuclear versus cytoplasmic isoforms that comprise the holoenzyme (9). Nuclear isoforms containing an NLS (CaMKII␣B, CaMKII␦3, and CaMKII␥A) could coassemble with cytoplasmic subunits, including postsynaptic density-associated CaMKII␣ (55) and/or F-actin-associated CaMKII␤ (56). Therefore, our results support the idea that assembly of CaMKII isoforms possibly affects nuclear translocation and activation.  Other studies have reported nuclear activity of other CaMKII isoforms, CaMKII␣B and CaMKII␥A, in neurons (57,58). For example, CaMKII␣B expression and nuclear translocation increase via an unknown mechanism following glutamate-induced cell death in rat retinal ganglion cells (57). CaMKII␣B knockdown also decreases neuronal BDNF expression (56). Ma et al. (58) have also reported that CaMKII␥A functions as a transporter of Ca 2ϩ /CaM to the nucleus following depolarization of cultured superior cervical ganglion neurons and that the Ca 2ϩ ⅐CaM-CaMKII␥ complex is dephosphorylated at Ser 334 by calcineurin, shuttling it to the nucleus. Nuclear delivery of Ca 2ϩ /CaM activates nuclear CaM kinases, including CaMKIV and CaMKK, driving CREB phosphorylation and transcription of CRE-regulated target genes (58). Therefore, phosphatases other than PP1, such as calcineurin and/or PP2A, may dephosphorylate at Ser 332 in other types of neurons.
We report here that chronic APZ treatment significantly increased BDNF protein expression concomitant with nuclear CaMKII␦3 translocation. APZ treatment also enhanced sprouting and survival of cultured dopaminergic neurons through the CaMKII␦3/PP1 pathway. Consistent with our results, APZ treatment for 8 weeks reportedly significantly increased plasma BDNF levels in first-episode untreated schizophrenia patients (59). APZ treatment also increases the pool size of long 3Ј-UTR Bdnf transcripts in the rat ventral hippocampus (60). Bdnf mRNAs carrying this type of UTR undergo dendritic targeting, and dendritically synthesized BDNF protein functions in dendritic morphogenesis (61). This evidence indicates that enhanced expression of BDNF protein following APZ treatment may represent a means to enhance the availability of Bdnf mRNA transcripts in dendrites, not only in nuclei, stimulating neurite extension. BDNF protein expression decreases in the dopamine-deficient substantia nigra of Parkinson disease patients (62,63). BDNF also reportedly promotes the survival of cultured mesencephalic dopaminergic neurons (64) and, in vivo, protects dopaminergic neurons from damage by the neurotoxins 1-methyl-1,2,3,6-tetrahydropiridine and 6-hydroxydopamine (65). In addition, D 2 R agonists have neuroprotective effects on various neurons in situ (54,66,67). For example, cabergoline blocks oxygen/glucose deprivation-induced cell death in SH-SY5Y neuroblastoma cells (66). In vivo, chronic cabergoline treatment antagonizes the death of dopaminergic neurons in 6-hydroxydopamine-treated mice (67). Because APZ is used clinically as a common prescription drug in schizophrenia, bipolar disorder, and depression, we selected APZ to define the mechanism underlying its neuroprotective effect. Although we have no data regarding whether quinpirole, another D 2 R agonist, has APZ-like effects on CaMKII␦3 dephosphorylation and nuclear translocation, quinpirole elicits neuroprotection against glutamate-induced neurotoxicity in cultured rat mesencephalic neurons (54). In addition, we have documented previously that stimulation with quinpirole in D 2 R-expressed NG108-15 cells activate the nuclear isoform of CaMKII (25). This evidence and our data suggest a critical role for BDNF in supporting the survival of midbrain dopaminergic neurons, an activity likely supported by the D 2 R-mediated CaMKII␦3/PP1 pathway.
Author Contributions-N. S. and K. F. conceived and coordinated the study and wrote the paper. N. S. and M. S. designed, performed, and analyzed the experiments shown in all figures. Y. I. and T. S. provided technical assistance. All authors reviewed the results and approved the final version of the manuscript.