A tardive dyskinesia drug target VMAT-2 participates in neuronal process elongation

Abstract

Tardive dyskinesia involves involuntary movements of body parts and is often observed in individuals taking antipsychotics for extended periods. Initial treatment strategies include reducing medication dosage, switching medications, or using drugs to suppress symptoms. One of the therapeutic targets for tardive dyskinesia is vesicular monoamine transporter-2 (VMAT-2, also known as solute carrier family 18 member A2 [SLC18A2]), which functions as an energy-dependent transporter of monoamines. The therapeutic drugs are used during adulthood, when neurons are maturing. For the first time, we report that treatment with a chemical VMAT-2 inhibitor reduces neuronal process elongation, a phenomenon commonly observed during development. Treatment with the inhibitors reserpine or tetrabenazine decreased process elongation in primary cortical neurons, and similar results were obtained in N1E-115 neuronal model cells undergoing process elongation. Knockdown of VMAT-2 using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas13-fitted guide RNA also reduced process elongation. However, treatment with reserpine or tetrabenazine did not affect the morphology of mature processes. Notably, treatment with hesperetin, a citrus flavonoid with neuroprotective effects, was able to restore the reduced process elongation induced by these inhibitors or VMAT-2 knockdown. The underlying molecular mechanism appeared to involve neuronal differentiation-related Akt kinase signaling. These results suggest that VMAT-2, as a drug target for tardive dyskinesia, plays a key role in process elongation and that some inhibitory effects of VMAT-2-targeted drugs on its elongation may be mitigated by co-administering a neuroprotective molecule.

Keywords: CRISPR-Cas13; Elongation; Hesperetin; Reserpine; Tardive dyskinesia; Tetrabenazine; VMAT-2.

Fig. 1

Reserpine does not affect process length after long-term culture following the induction of differentiation. (A, B) At 7 days after the induction of differentiation, N1E-115 cells were treated with reserpine or its vehicle and cultured for an additional 3 days. The morphology of the cells at day 0 is also depicted in the diagram. Cells with processes were counted and graphically represented.

Fig. 2

Tetrabenazine does not affect process length after long-term culture following the induction of differentiation. (A, B) At 7 days following the induction of differentiation, N1E-115 cells were treated with tetrabenazine or its vehicle and cultured for an additional 3 days. The morphology of the cells at day 0 is also depicted in the diagram. Cells with processes were counted and graphically represented.

Fig. 3

Treatment with reserpine leads to decreased process elongation. (A, B) Following the induction of differentiation, N1E-115 cells were treated with reserpine or its vehicle and cultured for 3 days. The morphology of the cells at day 0 is also depicted in the diagram. Cells with processes were counted and graphically represented (** p < 0.01; n = 10 fields). (C, D) The lysates of cells treated with reserpine or its vehicle were used for immunoblotting with antibodies against GAP43, Tau, and control actin. The expression levels of GAP43 and Tau were normalized to those of actin and graphically represented (** p < 0.01; n = 3 blots).

Fig. 4

Treatment with tetrabenazine leads to decreased process elongation. (A, B) Following the induction of differentiation, N1E-115 cells were treated with tetrabenazine or its vehicle and cultured for 3 days. The morphology of the cells at day 0 is also depicted in the diagram. Cells with processes were counted and graphically represented (** p < 0.01; n = 10 fields). (C, D) The lysates of cells treated with tetrabenazine or its vehicle were used for immunoblotting with antibodies against GAP43, Tau, and control actin. The expression levels of GAP43 and Tau were normalized to those of actin and graphically represented (** p < 0.01; n = 3 blots).

Fig. 5

Knockdown of VMAT-2 results in decreased process elongation. (A, B) N1E-115 cells were transfected with plasmids encoding Cas13 with control gRNA (gControl) or VMAT-2 one (gVMAT-2). Following the induction of differentiation, cells were cultured for 3 days. The morphology of the cells at day 0 is also depicted in the diagram. Cells with processes were counted and graphically represented (** p < 0.01; n = 10 fields). (C, D) The lysates of the respective cells were used for immunoblotting with antibodies against GAP43, Tau, and control actin. The expression levels of GAP43 and Tau were normalized to those of actin and graphically represented (** p < 0.01; n = 3 blots).

Fig. 6

Hesperetin recovers decreased process elongation under various experimental conditions. (A, B) Following the induction of differentiation, N1E-115 cells were treated with reserpine and hesperetin (Hes) or its vehicle and cultured for 3 days. The morphology of the cells at day 0 is also depicted in the diagram. Cells with processes were counted and graphically represented (** p < 0.01; n = 10 fields). (C, D) Following the induction of differentiation, cells were treated with tetrabenazine and hesperetin or its vehicle and cultured for 3 days. The morphology of the cells at day 0 is also depicted in the diagram. Cells with processes were counted and graphically represented (** p < 0.01; n = 10 fields). (E, F) Following the induction of differentiation, cells transfected with Cas13 and VMAT-2 gRNA (gVMAT-2) were treated with hesperetin or its vehicle and cultured for 3 days. The morphology of the cells at day 0 is also depicted in the diagram. Cells with processes were counted and graphically represented (** p < 0.01; n = 10 fields).

Fig. 7

Hesperetin recovers decreased Akt phosphorylation under various experimental conditions. (A, B) Following the induction of differentiation, N1E-115 cells were treated with reserpine and hesperetin (Hes) or its vehicle and cultured for 3 days. The immuneprecipitates using antibodies against phosphorylated Akt (pAkt) and the lysates of cells were used for immunoblotting with antibodies against total Akt (Akt). The levels of Akt immunoprecipitated with an anti-phosphorylated Akt antibody were normalized to those of total Akt or actin and graphically represented (** p < 0.01; n = 3 blots). The immunoreactive levels of Akt were also normalized to those of actin. (C, D) Following the induction of differentiation, cells were treated with tetrabenazine and hesperetin (Hes) or its vehicle. The levels of phosphorylated Akt immunoprecipitates were normalized to those of total Akt or actin and graphically represented (** p < 0.01; n = 3 blots). The immunoreactive levels of Akt were also normalized to those of actin. (E, F) Cells were transfected with plasmids encoding Cas13 with and VMAT-2 one (gVMAT-2). Following the induction of differentiation, cells were treated with or without hesperetin (Hes). The levels of phosphorylated Akt immunoprecipitates were normalized to those of total Akt or actin and graphically represented (** p < 0.01; n = 3 blots). The immunoreactive levels of Akt were also normalized to those of actin.