Haloperidol induces the nuclear translocation of phosphatidylinositol 3'-kinase to disrupt Akt phosphorylation in PC12 cells

Abstract

Objective: The antipsychotic drug haloperidol (HAL) has been linked to apoptosis and to inhibition of prosurvival Akt signalling in pheochromocytoma (PC12) and neuronal cell cultures. However, the mechanism involved is unclear.

Methods: We used HAL to induce cytotoxicity in preneuronal PC12 cells. The expression and the subcellular localization of selected components of the PI3K-Akt survival cascade were monitored with standard biochemical approaches, such as subcellular fractionation, western blot analysis, gene transfer and fluorescence microscopy.

Results: PC12 cell stimulation with the epidermal growth factor (used as a control) results in normal processing of phosphatidylinositol 3'-kinase (PI3K)-Akt signalling (e.g., localization of PI3K to the plasma membrane and phosphorylation of Akt (Ser473). Surprisingly, HAL induces PI3K-generated phosphoinositol [phosphatidylinositol-3,4,5-triphosphate (PIP3), which conflicts with its ability to inhibit Akt. In fact, the production of PIP3s is nuclear, as assessed by the localized concentration of a fluorophore-tagged PIP3-targeting pleckstrin homology protein and a fluorophore-tagged substrate-trapping mutant of the phosphoinositide phosphatase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). However, phosphoinositide-dependent protein kinase 1 (PDK1, the activating kinase of Akt) does not colocalize to the nucleus with the PI3K complex. This effectively inactivates both cytoplasmic and nuclear pools of Akt.

Conclusion: The differential compartmentalization of effectors of the PI3K-PDK1-Akt pathway is a unique means by which HAL disrupts Akt functioning in PC12 cells.

Keywords: EGF; haloperidol; phospholipids.

Fig. 1: Haloperidol (HAL) induces PI3K activity in PC12 cells. Protein extracts from HAL-treated (125 μM, 24 h) extracts were immunoprecipitated for p85 and subjected to a p110 lipid kinase assay (A). The [γ-³²P]-labelled 3'-phosphatidylinositol lipid products are indicated by PI3P on a representative autoradiogram (numbers indicate means of 2 experiments). Corresponding cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), transferred to nitrocellulose and probed to determine the expression levels of p85, Gab1, Src homology-2 domain-containing protein-tyrosine phosphatase (SHP-2) (B). Levels of β-actin were used to demonstrate equal protein loading in both lanes. These same cell lysates were also immunoprecipitated for p85 or for SHP-2. The resolved immunocomplexes were probed for p85 and Gab1. A p85(Y688D) activated protein (C) or an SHP-2(C459S) dominant negative catalytically inactive protein (D) were overexpressed in PC12 cell cultures to determine their influence on HAL-induced (125 μM, 24-hour) toxicity, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction. Data are presented as mean, standard deviation; n = 6–8; ns: not significantly different from vehicle control; *p < 0.05 and ***p < 0.001 versus vehicle (vector) control.

Fig. 2: Haloperidol (HAL) is toxic to PC12 cells. HAL treatment (125 μM, 24 h) results in an increase in the number of dead (labelled red) PC12 cells and induces a “rounding up” of cells, a characteristic of cells undergoing apoptosis. Bar: 20 μm.

Fig. 3: Fluorescence localization of components of the PI3K pathway in epidermal growth factor (EGF)- and haloperidol (HAL)-treated PC12 cells. (A) The localization of overexpressed proteins was monitored with confocal microscopy in EGF- (10 ng/ml; 10 min) or HAL- (125 μM, 24 h) treated PC12 cell cultures. The localization of these proteins to the plasma membrane (indicated by the arrows; centre column) is evident in EGF-treated cultures, whereas a pool of these same proteins (with the exception of PDK1) relocalizes to the nucleus (indicated by arrows; right column) in HAL-treated cultures. All micrographs are depicted through the plane of the nucleus. (B) The phosphorylation of Ser473-Akt is enhanced in EGF-treated PC12 cells, but not in HAL-treated cells. β-Actin demonstrates equal protein loading. (C) HAL treatment results in the concentration of the PH-RFP construct in the nucleus as demonstrated by counterstaining with the nuclear marker, 4',6-diamidino-2-phenylindole. Bar: 5 μm.

Fig. 4: Haloperidol (HAL) induces the nuclear translocation of p110/PI3K and dephosphorylates Akt in specific subcellular compartments. PC12 cells were treated with HAL (125 μM, 24 h). The cells were then fractionated, and the nuclear and cytosolic fractions were examined for the distribution of p85, p110 (indicate by arrow), phosphoThr308- (indicated by arrow) and phosphoSer473-Akt, phosphoSer241-protein kinase-1, and poly(ADP-ribose) polymerase (which demonstrates fraction purity). CTL = control; PARP = poly(ADP-ribose) polymerase.

Fig. 5: Membrane-directed, constitutively active Akt diminishes the effects of haloperidol (HAL). (A) Constitutively active myristoylated (myr; membrane-directed) Akt (myr-Akt) and PDK1 (myr-PDK1) were overexpressed (24 h) in PC12 cell cultures. Their effect on the cytotoxicity of 125 mM of HAL was monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction. Data are presented as mean, standard deviation; n ≥ 6: ***p < 0.001 versus vehicle control; #p < 0.05 relative to vector (vec)/HAL. (B) Protein extracts from similarly treated cells were used to demonstrate overexpression of the respective myristoylated-proteins and to monitor the phosphorylation status of Thr308- and Ser473-Akt residues. PDK1 = phosphoinositide-dependent protein kinase1; endo = endogenous.