Haloperidol Affects Plasticity of Differentiated NG-108 Cells Through σ1R/IP3R1 Complex

Haloperidol is an antipsychotic agent that primarily acts as an antagonist of D2 dopamine receptors. Besides other receptor systems, it targets sigma 1 receptors (σ1Rs) and inositol 1,4,5-trisphosphate receptors (IP3Rs). Aim of this work was to investigate possible changes in IP3Rs and σ1Rs resulting from haloperidol treatment and to propose physiological consequences in differentiated NG-108 cells, i.e., effect on cellular plasticity. Haloperidol treatment resulted in up-regulation of both type 1 IP3Rs (IP3R1s) and σ1Rs at mRNA and protein levels. Haloperidol treatment did not alter expression of other types of IP3Rs. Calcium release from endoplasmic reticulum (ER) mediated by increased amount of IP3R1s elevated cytosolic calcium and generated ER stress. IP3R1s were bound to σ1Rs, and translocation of this complex from ER to nucleus occurred in the group of cells treated with haloperidol, which was followed by increased nuclear calcium levels. Haloperidol-induced changes in cytosolic, reticular, and nuclear calcium levels were similar when specific σ1 blocker -BD 1047- was used. Changes in calcium levels in nucleus, ER, and cytoplasm might be responsible for alterations in cellular plasticity, because length of neurites increased and number of neurites decreased in haloperidol-treated differentiated NG-108 cells.


Introduction
Haloperidol is a typical antipsychotic agent used in the treatment of psychiatric disorders, including various psychoses such as schizophrenia and severe agitated delirium.
Several adverse effects of haloperidol treatment are reported among them extrapyramidal side effects such as dystonia and muscle rigidity, palpitations, and changes of arterial blood pressure are common; QT interval prolongation eventually followed by cardiac arrhythmias such as Torsade de Pointe, are also reported (Remijnse et al. 2002).
Haloperidol exhibits high-affinity dopamine D2 receptor (D2R) antagonism. D2Rs play an important role in pathophysiology of brain signaling. These receptors exist as monomeric units, but they can also form oligomers. D2 receptors are associated with Gi proteins to inhibit production of the cAMP. Nevertheless, recently, it was suggested that an imbalance of D1R/D2R heteromers could be related to depressive symptoms in youngsters (Corrales et al. 2016). Putative D1/D2 receptor heterodimers have been suggested to regulate diacylglycerol and IP 3 signaling by activating Gq (Rashid et al. 2007). It appears that D1 and D2 receptors are both necessary for this pathway; thus, the application of dopamine or a combination of two selective D1 and D2 receptor agonists is able to increase intracellular calcium, whereas treatment with either D1 or D2 receptor antagonist can abolish this effect (Hasbi et al. 2009).
Haloperidol is also known as a ligand of type 1 sigma receptors (r1Rs). The r1Rs were first discovered in the central nervous system (Martin et al. 1976) and later, their presence was shown in various tissues (Su and Junien 1994), including heart muscle (Dumont and Lemaire 1991;Novakova et al. 1995). The r1Rs are non-opioid transmembrane proteins located at the ER, mitochondrial, and plasma membranes (Hayashi and Su 2007). Several in vivo and in vitro studies have shown that overexpression of the r1R or activation of r1R by high-affinity ligands protect against neuronal cell death (Martin et al. 2004;Bucolo et al. 2006;Dun et al. 2007;Smith et al. 2008;Zhang et al. 2011).  reported that in vitro r1R ligands regulate levels of intracellular Ca 2? concomitantly with the attenuated activation of pro-apoptotic genes. Increasing r1R in vitro counteracts the ER stress response, whereas decreasing r1R enhances apoptosis (Hayashi and Su 2007). Upon ER-Ca 2? depletion or ligand stimulation, r1Rs dissociate from BiP/GRP78, leading to prolonged Ca 2? signaling into the mitochondria via inositol IP 3 Rs. Previously, we have shown that in isolated rat cardiomyocytes, r1Rs are coupled to type 1 and type 2 IP 3 Rs (Novakova et al. 2007), since silencing of these receptors attenuated expression of the r1R. Type 3 IP 3 R is also associated with r1R (Hayashi and Su 2001). It has been proposed that in this complex, r1R protects IP 3 R from degradation, whereas IP 3 R facilitates the transfer of calcium into the mitochondria and favors cell survival (Kiviluoto et al. 2013).
Because r1Rs bind to a broad range of synthetic compounds including antipsychotics, they are thought to be potential therapeutic targets for mental disorders; furthermore, r1Rs might play a pivotal role in neuroprotection (Hayashi and Su 2007;Katnik et al. 2006). Mitsuda and coworkers (2011) have shown that a transcription factor, ATF4, which is considered to be a marker of ER stress, directly binds to the 5 0 upstream region of r1R and modulates its expression. Additionally, the knock-down of ATF4 results in a decrease in the level of r1R expression. Thus, ER stress, which deeply involves IP 3 Rs, is likely to be a potent modulator of r1Rs acting through the ATF4 transcription factor.
We hypothesized that haloperidol might affect plasticity of neuronal cells by modulating predominantly r1Rs and IP 3 Rs, but also D2 receptors. As a model of neuronal cells we used NG-108 stable cell line differentiated by cAMP to the neuronal phenotype (Kubickova et al. 2016). We proposed that mutual interaction/communication of these three receptors in the presence of haloperidol might alter calcium fluxes and change the plasticity of differentiated NG-108 cells.

Cell Culture
The neuroblastoma-glioma cell line NG-108 (PAA Laboratories, Germany, provided by Dr. Lacinova) was used in these experiments. This line was formed by fusing mouse N18TG2 neuroblastoma cells with rat C6-BU-1 glioma cells in the presence of inactivated Sendai virus (Hamprecht 1977). Cells were plated at relatively low density (0.65 9 10 4 cells/cm 2 ), cultivated for 24 h and differentiated with dibutyryl cAMP (dbcAMP; Sigma, USA) as described in Kubickova et al. (2016). After differentiation, these cells are accepted as a model of neuronal cells.

Western Blot Analysis
Protein concentration of the lysate was determined by using the method of Lowry (1951). Whole procedure is described in detail in Lencesova et al. (2013). An enhanced chemiluminescence detection system (Luminata TM Crescendo Western HRP Substrate, Millipore) was used to detect the bound antibodies, and the optical density of individual bands was quantified using PCBAS 2.0 software.
To detect r1R protein, we used a rabbit polyclonal antibody against OPRS1 (AB_881796, Abcam, UK), a synthetic peptide derived from the C-terminal region of rat r1R peptide that recognizes a band of approximately 25 kDa. To detect IP 3 R1 protein, we used a rabbit polyclonal antibody derived from amino acids 1829-1848 of the cytoplasmic C-terminal domain of human IP 3 R1 (AB_260119, Sigma, USA), which recognizes a band of approximately 240 kDa. This sequence is 100% conserved in human, mouse, and rat IP 3 R1.

Immunoprecipitation
The appropriate monoclonal (3 lg) or polyclonal antibody (6 lg) was incubated with 60 ll of washed magnetic beads (Dynabeads M-280 coated with sheep anti-mouse IgG or M-280 coated with sheep anti-rabbit IgG (Life Technologies, Dynal AS, Norway)) overnight at 4°C on a rotator (VWR International, LLC, PA, USA). The beads with attached antibodies were washed twice (200 ll) with phosphate-buffered saline (PBS supplemented with 1% bovine serum albumin). Proteins were immunoprecipitated from 1 mg of detergent-extracted total protein via their incubation with antibody-bound beads for 4 h at 4°C. Bead complexes were washed with PTA (49 with 200 ll; 145 mmol/L NaCl, 10 mmol/L NaH 2 PO 4 , 10 mmol/L sodium azide, and 0.5% Tween 20; pH 7.0). Immunoprecipitated proteins were then extracted with 60 ll of 29 Laemmli loading buffer according to the manufacturer's instructions (Bio-Rad) and boiled for 5 min. The following antibodies were used for immunoprecipitation: rabbit polyclonal antibody to OPRS1 (r1R; AB_881796, Abcam, UK) and mouse monoclonal antibody to IP 3 R1 (AB_212025, Calbiochem, Merck Biosciences, Germany).

Immunofluorescence
Cells grown on glass coverslips were fixed in ice-cold methanol. Nonspecific binding was blocked by incubation with PBS containing 3% bovine serum albumin (BSA) for 60 min at 37°C. The cells were then incubated with primary antibody diluted 1:500 in PBS with 1% BSA (PBS-BSA) for 1 h at 37°C. A rabbit polyclonal antibody (AB_212026, Calbiochem, Merck Biosciences, Darmstadt, Germany) directed against 1829-1848 amino acid residues from human IP 3 R1 was used. Another group of cells was incubated with rabbit polyclonal antibody anti-OPRS1 (AB_881796, Abcam, USA) directed against a synthetic peptide derived from the C-terminal region of rat r1 peptide. Afterwards, the cells were washed three times with PBS/BSA for 10 min, incubated with CF488A goat antirabbit IgG (AB_10559670, Biotium) diluted 1:1000 in PBS/BSA for 1 h at 37°C, and washed as described previously. Finally, the cells were mounted onto slides in mounting medium with Citifluor (Agar Scientific Ltd., Essex, UK) and analyzed by laser scanning confocal microscopy (LSM 510 MetaMicroscope, Zeiss). Images were taken with a Plan Neofluar 409/1.3 oil objective. Images were scanned at scan speed 7 (260 Hz line frequency), 1024 9 1024 pixels, 12 bit data depth in the average mode (49 line) at optical zoom 3. The Z-stack interval was 0.8 lm. Images of all samples were acquired with the same microscope setup.

Proximity Ligation Assay (PLA)
PLA was used for the in situ detection of the interaction between D1 and D2 receptors and also between r1Rs and IP 3 R1s. The assay was performed in a humidified chamber at 37°C according to the instructions of the manufacturer (Olink Bioscience, Sweden). For this method, following antibodies were used: rabbit polyclonal antibody to OPRS1 (r1R; AB_881796, Abcam, UK), mouse monoclonal antibody to IP 3 R1 (AB_212025, Calbiochem, Merck Biosciences, Germany), mouse monoclonal antibody to dopamine receptor D1 (SG2-D1a, ab78021, Abcam, UK), and rabbit polyclonal antibody to dopamine receptor D2 (ab21218, Abcam, UK).

Cytosolic [Ca 21 ] i Staining by Fluo-3AM Fluorescent Dye
For this method we used a fluorescent dye Fluo-3AM (Sigma Aldrich, USA). Method is described in detail in Kubickova et al. (2016).

Determination of Reticular Calcium by Rhod-5 N
A detailed protocol has been described by (Lencesova et al. 2013). Rhod-5 N fluorescent dye (Invitrogen Ltd., Paisley, UK) was added to each sample to a final concentration 20 lmol/L, and measurements were taken using a BioTek fluorescent reader (excitation 551 nm/emission 576 nm). The results are expressed in arbitrary units.

Determination of Nuclear Calcium by Rhod-5 N
After 24 h of treatment, cells were gently collected from flasks, allowed to settle, and washed with 19 PBS solution. Gentle lysis was performed with 500 ll of cell lysis buffer from a kit for cytoplasmic and nuclear protein isolation (ProteoJetTM Fermentas, Germany) and 1,4-dithiothreitol to a final concentration of 1 mmol/L. The isolation of cell nuclei was performed according to the kit manufacturer's instructions. Pellets from the nuclear fraction were homogenized in 200 ll of nuclear lysis buffer from the ProteoJetTM kit and pipetted into a 24-well plate. For each sample, Rhod-5 N fluorescent dye was added to a final concentration of 20 lmol/L, and measurements were taken using a BioTec fluorescent reader (BioTec, Germany) at 551 nm (excitation) and 576 nm (emission). After the fluorescence was measured, the signal was quenched by adding EGTA solution (pH 7.0) to final concentrations of 0.25, 1.0, 2.5, and 5.0 mmol/L. The results are expressed in arbitrary units.

Quantification of Neurite's Outgrowth
Neurite outgrowth was determined as described in Kubickova et al. (2016). Quantification of neurite outgrowth was verified by ''Neurite Outgrowth Staining Kit''. To visualize cell viability and neurite's outgrowth we used a dual-color stain (Life Technologies, Dynal AS, Norway). For our experiments, we used combination of the cell viability indicator and the cell membrane stain (diluted 1000-fold) in Dulbecco's Phosphate-Buffered Saline (DPBS, Thermo Fisher Scientific, Hampshire, UK) containing calcium and magnesium. Neurite outgrowth was analyzed by laser scanning confocal microscopy (LSM 510 MetaMicroscope, Zeiss) and also by BioTek fluorescence scanner (BioTek, Germany), where quantification of a relative fluorescence was performed. Indicator of cell viability was measured using excitation/emission wavelengths of 483/525 nm and cell membrane stain was measured at excitation/emission wavelengths of 554/567 nm. The results were expressed as arbitrary units.

Statistical Analysis
Each value represents an average of 3-9 wells from at least two independent cultivations of NG-108 cells. The results are presented as the mean ± S.E.M. Significant differences between the groups were determined by one-way ANOVA. For multiple comparisons, an adjusted t test with p values corrected by the Bonferroni method was used.

Results
In differentiated NG-108 cells, we observed a concentration-dependent increase in IP 3 R1 mRNA ( Fig. 1a; black columns) and in r1R ( Fig. 1b; black columns), while in non-differentiated cells, no changes in the corresponding mRNA ( Fig. 1a, b; striped columns) or protein (Fig. 1c, d) were visible. In differentiated cells, treatment with haloperidol at a concentration of 10 nmol/L (Hn) for 24 h increased IP 3 R1 mRNA levels from 1.0 ± 0.4 a.u. to 2.7 ± 0.1 a.u. (**p \ 0.01), while the mRNA levels of r1R were increased by haloperidol treatment (from 1.0 ± 0.1 a.u. to 1.9 ± 0.2 a.u., **p \ 0.01) only at the concentration of 10 lmol/L (Hl). Additionally, we observed a significant increase in protein expression of IP 3 R1 (Fig. 1c) and r1R (Fig. 1d) after 24 h of Hl treatment. The expression of the type 3 IP 3 R was unchanged following this treatment (Fig. 1f). IP 3 R2s are not expressed in differentiated NG-108 cells (Fig. 1e), as verified using the PC12 cell type, where a clear signal of the IP 3 R2 was visible. We proposed that increased level of IP 3 R1 due to Hl treatment might be responsible for increased levels of cytosolic calcium. Therefore, we silenced IP 3 R1, IP 3 R3, or combination of both and determined levels of cytosolic calcium with/without Hl treatment (Fig. 2a). Silencing of the IP 3 R1 or IP 3 R1/IP 3 R3 followed by Hl treatment resulted in decreased levels of cytosolic calcium, thus proving involvement of this receptor in Hl-induced increase of cytosolic calcium (Fig. 2a). Silencing of the IP 3 R3 and Hl treatment did not change calcium levels compared to Hl treated group. In control cells, the IP 3 R1 was localized to the endoplasmic reticulum, but after Hl treatment; we observed the translocation of IP 3 R1 from the ER to the nucleus ( Fig. 2b; green sinal) and translocation of r1Rs to the nucleus as well ( Fig. 2c; green signal). To further verify the translocation of IP 3 R1 and r1Rs, we obtained confocal z-stacks from the images that confirmed a positive signal in the nucleus (Fig. 2d). Following simultaneous incubation with Hl and Xest (1 lmol/L), r1Rs remain localized primarily to the ER (Fig. 2e). Colocalization of IP 3 R1 with r1Rs was determined by proximity ligation assay (Fig. 3a) and immunoprecipitation (Fig. 3b). By immunoprecipitation, we clearly showed that IP 3 R1 co-immunoprecipitates with r1Rs ( Fig. 3b; left) in control cells and in Hl and BDl-treated cells. Reverse immunoprecipitation with IP 3 R1 resulted in the co-immunoprecipitation of r1Rs ( Fig. 3b; right), further demonstrating the clustering of these receptors. Negative controls verified the specificity of the immunoprecipitation. This observation was verified by a proximity ligation assay, where red dots showing the interaction of these two receptors were observed (Fig. 3a). Since haloperidol is a nonspecific ligand of r1Rs, we compared the results observed following haloperidol treatment with those from a specific blocker of r1Rs, BD 1047 at a concentration of Fig. 1 Haloperidol increases the mRNA (a, b) and protein (c, d) levels of r1R (b, d) and type 1 (a, c), but not type 2 (e) and 3 (f), IP 3 receptors in differentiated NG-108 cells (Dif). In contrast to nondifferentiated (ND) cells (a, b striped columns), in differentiated cells (a, b black columns) haloperidol at a concentration of 10 nmol/L (low-dose; Hn) increases the mRNA expression of IP 3 R1, and at a concentration of 10 lmol/L (high-dose; Hl), the mRNA and protein expression of both IP 3 R1 and rR1 was increased (a, b). Type 2 IP 3 receptors are not expressed in NG-108 cells (e); we observed an expression signal in PC12 cells but not in NG-108 cells. The mRNA expression of type 3 IP 3 receptors was not changed in undifferentiated or in differentiated cells following Hl treatment (f). The results are expressed as the mean ± SEM. Statistical significance: **p \ 0.01 and ***p \ 0.0001 compared to control untreated cells Cell Mol Neurobiol (2018) 38:181-194 185 10 lmol/L (BDl). Western blot analysis documented the higher amount of r1R protein in Hl treated cells and BDl treated cells compared to untreated control cells (Fig. 2e). A significant increase in cytosolic calcium was observed in Hl-treated cells compared to untreated controls ( Fig. 4a; from 933 ± 14 a.u. to 1323 ± 64 a.u., ***p \ 0.0001). A rapid decrease in cytosolic calcium occurs when cells were treated with both Hl and the IP 3 R blocker Xest (567 ± 48 a.u., ???p \ 0.0001). Also, we observed a significant increase in cytosolic calcium following treatment with BDl and a rapid decrease when cells were treated in parallel with BDl and Xest ( Fig. 4a; from 1070 ± 27 a.u. to 421 ± 10 a.u., ???p \ 0.0001). Involvement of r1Rs in the Hl-induced increase of the cytosolic calcium was verified by r1Rs silencing using appropriate siRNA (Fig. 4b). Hl-induced increase of the cytosolic calcium level was prevented also by a parallel treatment with Xest or a specific r1Rs agonist SA4503 (1 lmol/L) (Fig. 4b).
Modulation of cytosolic calcium by the IP 3 R blocker Xest suggests a release of reticular calcium stores. Thus, we measured reticular calcium in Hl and BDl-treated cells (Fig. 4c) and we observed that in both cases, the level of reticular calcium decreased following a 24-h treatment (from 651.0 ± 12.8 a.u. to 500.2 ± 10.8 a.u. (Hl), **p \ 0.01; or 617.4 ± 21.3 a.u. (BDl), and was significantly increased when Xest was added in parallel Fig. 2 Involvement of IP 3 R1, but not IP 3 R3 in haloperidol-induced changes in levels of cytosolic calcium and translocation of the IP 3 R1 and r1R to the nucleus following haloperidol treatment. Experiments were performed on NG-108 cells differentiated by dbcAMP. To determined haloperidol-induced changes in cytosolic calcium due to IP 3 R1/IP 3 R3 receptors, these receptors were silenced either individually, or both of them and levels of cytosolic calcium levels were determined after haloperidol treatment (a). Silencing of IP 3 R1, but not IP 3 R3 caused significant decrease in cytosolic calcium levels, thus proving involvement of the IP 3 R1, but not IP 3 R3 in haloperidolinduced increase in cytosolic calcium. In control cells (cont), IP 3 R1s (b, green signal) and r1Rs (c, green signal) are localized to the ER. Following haloperidol treatment (Hl; 10 lmol/L), these receptors translocate to the nucleus (b, c, green signal). Translocation of the IP 3 R1 and r1R was verified by z-stacks from the Hl-treated cells (d), which clearly shows an intranuclear signal. In the presence of Xestospongin C (Xest; 1 lmol/L), the Hl -induced translocation of r1R does not occur (e, HlXest). Results in the graph are expressed as the mean ± SEM and represent an average of six parallels from two independent cultivations. Statistical significance compared to control was ***p \ 0.0001 and compared to Hl treated cells was ???p \ 0.0001 (785.1 ± 40.4 a.u. (Hl); or 765.3 ± 15.0 a.u. (BDl)). Silencing of the r1Rs mRNA in Hl-treated cells resulted in an increase of the reticular calcium, similarly as SA4503 (Fig. 4d). Interestingly, huge increase in reticular calcium compared to untreated cells occurs, when Hl-treated cells were incubated in parallel with both, Xest and SA4503 (Fig. 4d). Because Hl treatment results in the translocation of both IP 3 R1 and r1Rs to the nucleus, we measured nuclear calcium levels in isolated nuclei (Fig. 4e). Both Hl and BDl treatments significantly increased the level of nuclear calcium after 24 h (from 73.5 ± 1.1 a.u. to 345.5 ± 3.9 a.u. (Hl), ***p \ 0.0001; or 151.0 ± 2.1 a.u. (BDl), *p \ 0.05). Xest decreased the level of nuclear calcium in Hl-treated cells but surprisingly led to a rapid increase in nuclear calcium levels in BDl-treated cells ( Fig. 4e; 139.6 ± 29.0 a.u. (Hl); or 645.8 ± 6.6 a.u. (BDl), ???p \ 0.0001). In Hl-treated cells, silencing of the r1Rs mRNA significantly decreased a level of nuclear calcium compared to plain Hl-treated cells (Fig. 4f).
The physiological impact of these treatments was determined by measuring the number of neurites per cell and neurite outgrowth in cells treated with haloperidol and BD 1047 along with those treatments in combination with the Xest. Haloperidol (H10 -7 -H10 -4 ) treatment decreased the number of neurites in a concentration-dependent manner ( Fig. 5a). However, length of neurites increased due to a haloperidol treatment in a concentration-dependent manner. This increase was prevented, when haloperidol-treated cells were incubated with Xest in parallel (HXest; Fig. 5b). Similar results were obtained when BD 1047 (BD10 -7 -BD10 -4 ) was used (Fig. 5c, d). However, effect of the BD 1047/Xest treatment (BDXest) on the neurite's outgrowth was highly dependent on a BD 1047 concentration (Fig. 5d). Neither r1Rs agonist PRE-084 (PRE10 -7 -PRE10 -4 ), nor IP 3 R blocker Xest (Xest10 -8 -Xest10 -5 ) modulated length of neurites by a concentration-dependent manner (Fig. 5e, f).
Neurite outgrowth was measured in NG-108 cells differentiated for 72 h and further treated with Hl, BDl, and/or Xest, but also with the r1Rs agonists SA4503 and PRE-084 (10 lmol/L) (Fig. 6). For these measurements, dual approach was used-measuring of individual neurites (Fig. 6a) and evaluation of the fluorescence signal (Fig. 6b, c, d). We observed a significant increase in the length of neurites in Hland BDl-treated differentiated cells (Fig. 6a, b) compared to untreated control cells. Parallel treatment with Xest decreased partially the length of neurites (Fig. 6a, b). Elevated neurite outgrowth was clearly visible in Hl and BDl treated cells compared to control cells, or SA4503 and/or PRE-084 treated cells (Fig. 6c, d; red signal). Green signal shows the viability of cells (Fig. 6d). In order to show the participation of the r1Rs in Fig. 3 Interaction of the IP 3 R1 and r1R receptor was verified by proximity ligation assay and immunoprecipitation of these receptors. Experiments were performed on NG-108 cells differentiated by dbcAMP. The mutual interaction of the IP 3 R1 and r1R was verified by a proximity ligation assay (a), where red dots show co-localization of these two receptors. Bar represents 20 lm. NCI negative control without IP 3 R1 primary antibody, NCS negative control without r1R primary antibody. Immunoprecipitated r1Rs bound the IP 3 R1 in control cells (b; cont) and in cells treated with Hl or BDl (b). In agreement, immunoprecipitated IP 3 R1 bound r1Rs in Hl-and BDltreated cells (b). Western blot analysis documented amount of the r1R protein in control cells, Hl treated cells and BD 1047 (BDl; 10 lmol/L) treated cells (c) Cell Mol Neurobiol (2018) 38:181-194 187 the cell plasticity, we silenced these receptors and subsequently measure the number and length of neurites (Fig. 7). Silencing of the r1Rs in the Hl-treated cells significantly downregulates number and also length of neurites compared to Hl-treated cells with or without scrambled siRNA (Fig. 7a, b). Effectivity of the r1Rs silencing is visible on cell images ( Fig. 7c; green signal). Hl treatment increased markers of ER stress, CHOP (contr. 5.2 ± 0.1 a.u.; Hl 13.3 ± 1.6 a.u., ***p \ 0.0001), XBP1 (contr. 7.5 ± 0.2 a.u.; Hl 15.4 ± 1.8 a.u., **p \ 0.01), and ATF4 (contr. 5.6 ± 1.2 a.u.; Hl 9.7 ± 0.1 a.u., **p \ 0.01), in differentiated NG-108 cells (Fig. 7d).
This increase was also observed when a specific blocker of r1R, BDl, was used. Moreover, silencing of the r1Rs results in an increase of the gene expression of ER stress markers-CHOP, XBP1, and ATF4 (Fig. 7d). Parallel treatment with Xest partially prevented Hl induced gene expression of CHOP (Hl 13.3 ± 1.6 a.u.; Hl/Xest 6.6 ± 0.1 a.u.) or XBP1 (Hl 15.4 ± 1.8 a.u.; Hl/Xest 11.8 ± 0.6 a.u.). Unexpectedly, parallel treatment with Hl and Xest revealed the same ATF4 mRNA levels (Hl 9.7 ± 0.1 a.u.; Hl/Xest 9.7 ± 1.2) a.u. as in Hl treated cells (Fig. 7d). (1 lmol/L) and also with silenced r1R was used (b, d, f). As a control serves scrambled siRNA (scr). Hl treatment significantly increased the levels of cytosolic calcium, while Xest treatment in combination with Hl completely prevented this increase (a). Silencing of the r1R in Hl-treated cells significantly decreased a level of cytosolic calcium compared to scr or plain Hl-treated cells (b). The r1R-agonist SA4503 further decreased a level of cytosolic calcium compared to control cells (b). Accordingly, reticular calcium was decreased in Hl-treated cells compared to control cells, but Xest, SA4503 treatment, or silencing of the r1R increased a level of reticular calcium compared to Hl-treated cells (c, d). The results observed following BDl treatment were similar to those following Hl treatment. In nuclei, Hl increased nuclear calcium level, which was decreased by Xest, SA4503, or silencing of the r1Rs (e, f). Surprisingly, when cells were treated in parallel with BDl and Xest, huge increase in nuclear calcium level was observed compared to plain BDl-treated cells (e). Each column represents an average of six independent cultivations and is displayed as the mean ± S.E.M. Statistical significance compared to controls is * p \ 0.05, ** p \ 0.01, *** p \ 0.001, and compared to the haloperidol group is ??p \ 0.01 and ???p \ 0.001 It is known that signaling of D2 receptors is realized through Gi and inhibition of adenylate cyclase, while D1/ D2 heterodimeric complex acts through Gq and phospholipase C, which results in the IP 3 production. Using proximity ligation assay we observed clear co-localization of D1/D2 receptors in differentiated NG-108 control cells ( Fig. 8; red dots), but not in haloperidol-treated cells. These results suggest haloperidol-induced disintegration of D1/D2 receptor complex and thus switch from the IP 3 to cAMP signaling.

Discussion
In this work, we have clearly shown that in differentiated NG-108 cells haloperidol modulates plasticity of these cells, i.e., decreases number of neurites and increases the length of neurites. Haloperidol-induced changes in cell's plasticity are probably due to changes in cytosolic and reticular calcium that is modulated by up-regulation of the expression of IP 3 R1. Haloperidol increases expression of both IP 3 R1 and r1R in differentiated NG-108 cells. Since haloperidol also increases the expression of IP 3 R2s in cardiac atria (Novakova et al. 2010;Tagashira et al. 2013), we investigated the gene expression of type 2 and 3 IP 3 Rs in differentiated NG-108 cells. We observed that these cells do not express IP 3 R2s and that the expression of IP 3 R3s was unaffected by Hl treatment. Therefore, we focused our interest on the IP 3 R1.
Haloperidol increased cytosolic calcium compared to untreated controls. This increase was abolished by IP 3 R blocker Xestospongin C, which was used in parallel with haloperidol. In agreement, reticular calcium was decreased in cells treated with haloperidol and this decrease was prevented by Xestospongin C. Based on these results, we concluded that haloperidol-induced increased cytosolic calcium is due to calcium depletion from the reticulum. Since haloperidol increased expression of the IP 3 R1 (and not IP 3 R3), we propose that depletion of the reticulum is through the IP 3 R1. Another question is how r1Rs contribute to this process. The r1R antagonist BD1047 increased levels of cytosolic calcium, but did not change reticular calcium levels. However, levels of the nuclear calcium were increased by the treatment with BD1047, although not to the same extent as with haloperidol treatment. These results (together with results from immunofluorescence and proximity ligation assay) would suggest that sigma-1 receptor blocking plays the role primarily in increasing levels of the IP 3 R1s (but not their activity, since reticular calcium was not changed by BD 1047 treatment) and their translocation to the nucleus. This is supported by experiments with silenced r1R and haloperidol treatment in parallel, where we have observed that in these cells, haloperidol-induced increase in the nuclear calcium was lower in cells, where r1R was silenced. Also, in the cells where r1R was silenced, reticular calcium overload was detectable (not shown).
Taken together, we hypothesized that while haloperidol affects expression and activity of the IP 3 R1s, r1Rs might be more responsible for their trafficking into the nucleus (which in turn might affect expression of the IP 3 R1).
We have shown that Hl treatment for 24 h causes increase in the expression, complex formation, and translocation of both IP 3 R1s and r1Rs to the nucleus. The IP 3 R1/r1Rs complex has already been reported in Fig. 6 Effect of haloperidol (Hl) and BD1047 (BDl) on the length of neurites. Experiments were performed on NG-108 cells differentiated by dbcAMP. The cells were treated for 24 h with Hl (10 lmol/ L), BDl (10 lmol/L) and Xest (1 lmol/L) after 72 h of differentiation. Length of neurites was measured either manually by ImageJ program (a), or using ''Neurite outgrowth staining kit'' (b-d). By both methods it is clearly shown that Hl increased the length of neurites and this increase is partially prevented by Xest. Similar increase in length of neurites was visible after the BDl treatment, although the effect of Xest was not so conclusive (a, b). Results from the confocal microscopy without a cell viability staining (c; bar represents 50 lm) or together with the cell viability stain (d; bar represents 100 lm) supported results from the fluorescent reader. Each column represents an average of 450-835 cells, and the results are displayed as the mean ± S.E.M. Statistical significance compared to controls is *p \ 0.05, **p \ 0.01, and ***p \ 0.001 vs. control and ?p \ 0.05; ??p \ 0.01, and ???p \ 0.001 vs. Hl or BDl-treated cells hepatocytes (Abou-Lovergne et al. 2011). Additionally, it has been shown that r1Rs affect Ca 2? signaling in NG-108 (Hayashi and Su 2001) and MCF-7 cells via the formation of a trimeric complex with ankyrin B and IP 3 R3. In our hands, IP 3 R3s were not affected by Hl treatment in NG-108 cells; therefore, we focused on the IP 3 R1/r1Rs complex.
Previously, we have shown that IP 3 R1s aggregate and form intranuclear clusters when cells are treated with certain pro-apoptotic agents (Lencesova et al. 2013;Ondrias et al. 2011). Miki andco-workers (2015) have found intranuclear aggregates of r1Rs with huntingtin, and they reported that r1R is involved in the degradation of intranuclear inclusions in a cellular model of Huntington's disease. Additionally, translocated r1Rs have been shown to co-localize partially with PML bodies, which are suggested to play a role in transcriptional regulation and nuclear protein sequestration (Spector 2006) and also apoptosis. We propose that translocation of the r1Rs and IP 3 R1s might alter transcription of certain genes through changes in intranuclear calcium. It has been shown that r1Rs are involved in the regulation of intracellular [Ca 2? ] i by affecting Ca 2? -influx Fig. 7 The impact of haloperidol treatment on cell's plasticity. Experiments were performed on NG-108 cells differentiated by dbcAMP. The pharmacological impact was determined by measuring the number of neurites per cell (a) and neurite outgrowth (b) in haloperidol (Hl; 10 lmol/L)-treated cells with a parallel treatment of Xestospongin C (Xest; 1 lmol/L), SA4503 (1 lmol/L) and with silenced r1R. Number of neurites decreased significantly in Hltreated cells with silenced r1R (a), but not with a scrambled siRNA. Silencing of the r1R in Hl-treated cells decreased significantly compared to plain Hl-treated cell, similarly as by r1R agonist SA4503 (b). Effectivity of r1R silencing was verified by immunofluorescent staining (c). Bar represents 20 lm. Induction of markers of ER stress in Hl, BDl, and siRNA r1R -treated differentiated NG-108 cells (d). The relative mRNA levels of CHOP, XBP1, and ATF4 were determined in control (cont), Hl-treated and BDl cells with or without Xest, then in cells after silencing of the r1R (silr1R) and scrambled siRNA (scr). A significant increase compared to control was observed in silr1R cells and in Hl-and BDl-treated cells, but not in combination of these compounds with Xest. The results are expressed as the mean ± SEM. Statistical significance: * p \ 0.05, ** p \ 0.01, and *** p \ 0.001 compared to untreated control cells; ? p \ 0.05, ?? p \ 0.01 compared to Hl, BD, and/or silr1R treated group Cell Mol Neurobiol (2018) 38:181-194 191 or the release from intracellular stores (Gasparre et al. 2012). The Ca 2? -response triggered by an extracellular ligand engaging the IP 3 /Ca 2? pathway can be increased by r1R agonists and decreased by r1R antagonists (Gasparre et al. 2012). The r1Rs could affect Ca 2? signaling because it has been shown that r1R ligands affect Ca 2? -influx and the beating rate of cardiac myocytes (Ela et al. 1994). We observed that Hl increased cytosolic calcium levels compared to control untreated cells. At the same time, a decrease in reticular calcium occurs suggesting the depletion of the ER via IP 3 R1. Calcium depletion in the ER is accompanied by ER stress. Indeed, we observed increased markers of ER stress such as ATF4, XBP1, and CHOP in haloperidol-treated group. Involvement of the r1Rs in ER stress was proved by their silencing and subsequent increased levels of abovementioned markers. In cancer cells, r1R antagonists evoke ER stress response that is inhibited by r1R agonists (Do et al. 2013;Mori et al. 2013;Wang et al. 2012). Omi and coworkers (2014) demonstrated that ER stress induces r1R expression through the PERK pathway, which is one of the cell's responses to ER stress. In addition, it has been demonstrated that induction of r1R can repress cell death signaling. Thus, we propose that ER stress might be a trigger for r1R overexpression, binding to the IP 3 R1s and translocation of this complex to the nuclei. Also, ER stress correlates with altered plasticity of NG-108 cells. Indeed, involvement of the ER stress in morphological changes of differentiated NG-108 cells was verified by ER stressor thapsigargin, which generated similar morphological changes as haloperidol (Kubickova et al. 2016). Finally, nuclear calcium was increased, which might be due to the translocation of IP 3 R1s to the nucleus. Mitsuda and co-workers (2011) have shown that r1Rs are transcriptionally upregulated via the PERK/eIF2a/ATF4 pathway and ameliorate cell death signaling. Miki and co-workers (2014) had reported that ER stress caused translocation of r1Rs from cytoplasm to the nucleus. Function of r1Rs in the nucleus should be further elucidated. Crottes and co-workers (2013) proposed functional consequences of such translocation. Because of the spatial dynamics of r1Rs within the cell, the protein could also behave as a transcription factor that directly or indirectly controls a set of genes that encode ion channels. Many reports have shown the involvement of r1R in a number of signaling pathways that potentially target transcriptional activity (e.g., MAP kinases, PKA, PI3 K/AKT, NFj-B, c-Fos, CREB) (Crottes et al. 2013). Another interesting issue is the mechanism of dopamine signaling. Haloperidol is primarily an antagonist of D2 receptors. These receptors generally transmit signal through Gi and inhibition of adenylyl cyclase. However, D1/D2 receptors can transmit signal through Gq and production of IP 3 (for review see Beaulieu et al. 2015). We observed that D1/D2 heterodimers really occurred in a control group of differentiated NG-108 cells, but not in a group treated with haloperidol. Based on these results, we propose that due to block of D2 receptors by haloperidol, disintegration of D1/D2 complex occurs, and activity of the IP 3 R1 is significantly decreased due to a lack of IP 3 . Therefore, cells start to increase the IP 3 R1 expression. On the other hand, treatment with the r1R antagonist BD 1047 did not lead to disintegration of D1/ D2 complex. This observation would support the protective role of r1Rs on IP 3 R1s, rather than its regulatory role.
An unexpected result was observed in our experiments. Nuclear calcium was significantly increased in BDl/Xesttreated cells compared to cells treated with BDl only. Additional experiments are needed to clarify this phenomenon. We propose that this change might be a compensatory mechanism involved in the regulation of transcription by r1Rs.
We measured the length of neurites in differentiated cells following a 24-h treatment with either haloperidol or BD 1047, or after silencing r1R. Haloperidol treatment for 24 h modulates the plasticity of differentiated NG-108 cells, and haloperidol and BD 1047 significantly increases Fig. 8 The effect of haloperidol treatment on disintegration of D1/D2 heterodimeric complex in differentiated NG-108 cells. Mutual interaction of the D1 and D2 receptor was verified by a proximity ligation assay, where red dots (marked also by arrows) show colocalization of these two receptors in control (Cont) and BD1047 treated cells (BDl), but not in haloperidol-treated (Hl) group. Bar represents 20 lm. NCD2 negative control without D2 receptor's primary antibody, NCD1 negative control without D1 receptor's primary antibody the length of neurites and decreases their numbers per cell. Our observation does not agree with that of Ishima and Hashimoto (2012), who have shown that the potentiation of NGF-induced neurite outgrowth mediated by ifenprodil (a prototypical antagonist of the N-methyl-D-aspartate receptor) was significantly antagonized by the co-administration of the selective r1R antagonist NE-100. The r1R activation has been shown to promote neurite outgrowth in cerebellar granule neurons through the phosphorylation of tropomyosin receptor kinase B at Y515 (Kimura et al. 2013). From these results and from the literature (Kimura et al. 2013;Ishima et al. 2014), it is clear that r1R affects cell plasticity. This demonstration of plasticity is dependent on the compound affecting the r1Rs, the time and length of exposure and the differentiation status of the cells. Rather controversial results of various studies might originate from different cell types, affinity of ligands to r1R, different concentrations of ligands used and methodology of neurite outgrowth assessment. Nevertheless, further studies on this issue are required.
In conclusion, haloperidol treatment causes disruption of D1/D2 heterodimer and suppression of the IP 3 R activity. This probably leads to an increase of IP 3 R1 expression, depletion of calcium from ER, which generates ER stress. As a consequence, r1Rs are also upregulated. Both IP3R1 and r1Rs form a cluster and translocate to the nucleus, where they increase the level of intranuclear calcium. In differentiated NG-108 cells, this process is likely to result in changes to neuronal plasticity.