Mirtazapine exerts astrocyte-mediated dopaminergic neuroprotection

Abstract

Mirtazapine, a noradrenergic and specific serotonergic antidepressant (NaSSA), is known to activate serotonin (5-HT) 1A receptor. Our recent study demonstrated that stimulation of astrocytic 5-HT1A receptors promoted astrocyte proliferation and upregulated antioxidative property in astrocytes to protect dopaminergic neurons against oxidative stress. Here, we evaluated the neuroprotective effects of mirtazapine against dopaminergic neurodegeneration in models of Parkinson's disease (PD). Mirtazapine administration attenuated the loss of dopaminergic neurons in the substantia nigra and increased the expression of the antioxidative molecule metallothionein (MT) in the striatal astrocytes of 6-hydroxydopamine (6-OHDA)-injected parkinsonian mice via 5-HT1A receptors. Mirtazapine protected dopaminergic neurons against 6-OHDA-induced neurotoxicity in mesencephalic neuron and striatal astrocyte cocultures, but not in enriched neuronal cultures. Mirtazapine-treated neuron-conditioned medium (Mir-NCM) induced astrocyte proliferation and upregulated MT expression via 5-HT1A receptors on astrocytes. Furthermore, treatment with medium from Mir-NCM-treated astrocytes protected dopaminergic neurons against 6-OHDA neurotoxicity, and these effects were attenuated by treatment with a MT-1/2-specific antibody or 5-HT1A antagonist. Our study suggests that mirtazapine could be an effective disease-modifying drug for PD and highlights that astrocytic 5-HT1A receptors may be a novel target for the treatment of PD.

Figure. 1

Mirtazapine administration ameliorates dopaminergic neurodegeneration via 5-HT1A receptor in parkinsonian mice. (a) Representative photomicrographs of TH immunohistochemistry in the SNpc of parkinsonian mice after administration of mirtazapine (5 or 16 mg/kg). Scale bar = 200 µm. (b) High magnification images of (a). Scale bar = 100 μm. (c) Changes in the number of TH-positive neurons after mirtazapine administration. (d) Representative photomicrographs of TH immunostaining in the SNpc of mirtazapine (16 mg/kg) and WAY100635 (WAY; 0.5 mg/kg) co-administered parkinsonian mice. Scale bar = 200 µm. (e) High magnification images of (d). Scale bar = 100 μm. (f) Quantification of the number of TH-positive cells. Data are presented as means ± SEM (n = 5–6 slices/group). *p < 0.05, **p < 0.01 and ***p < 0.001 versus control side of vehicle-treated group. ^## p < 0.01, ^###p < 0.001 between the two indicated groups.

Figure. 2

Mirtazapine administration promotes astrocyte proliferation and MT-1/2 upregulation in the striatal astrocytes of parkinsonian mice. (a,c,e) Representative photomicrographs of immunohistochemistry for S100β (green) and MT-1/2 (red) (a), GFAP (green) and MT-1/2 (red) (c), and MT-1/2 (red) (e) in the striatum of mirtazapine (5 or 16 mg/kg)-treated parkinsonian mice. Scale bar = 50 µm. (b) Quantification of the number of S100β- and MT-1/2-positive cells. (d) Quantification of the number of GFAP- and MT-1/2-positive cells. (f) Quantification of the optical density of MT-1/2-immunoreactivity. Data are presented as means ± SEM (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control side of each group. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus same side of vehicle-treated group. ⁺p < 0.05 between the two indicated groups.

Figure. 3

Mirtazapine promotes MT upregulation via 5-HT1A receptors. (a,c,e) Representative photomicrographs of immunohistochemistry for S100β (green) and MT-1/2 (red) (a), GFAP (green) and MT-1/2 (red) (c), and MT-1/2 (e) in the striatum of mirtazapine (16 mg/kg) and WAY100635 (WAY; 0.5 mg/kg) co-administered parkinsonian mice. Scale bar = 50 µm. (b) Quantification of the number of S100β- and MT-1/2-positive cells. (d) Quantification of the number of GFAP- and MT-1/2 positive cells. (f) Quantification of the optical density of MT-1/2-immunoreactivity. Data are presented as means ± SEM (n = 5–6). ^#p < 0.05 and ^##p < 0.01 versus lesioned side of vehicle-treated group. ^§§p < 0.01 and ^§§§p < 0.001 vs. mirtazapine (16 mg/kg)-treated group.

Figure. 4

Mirtazapine requires astrocytes to exert 5-HT1A receptor-mediated dopaminergic neuroprotection. (a) Changes in the number of TH-positive cells in enriched neuronal cultures after treatment with mirtazapine (10 µM) followed by exposure to 6-OHDA (10–50 µM). (b) Changes in the number of TH-positive cells in mesencephalic neuron and striatal astrocyte cocultures after treatment with mirtazapine (10 µM) followed by exposure to 6-OHDA (50–100 µM). (c) Changes in the number of TH-positive cells in neuron and astrocyte cocultures after treatment with mirtazapine (10 µM) and the 5-HT1A receptor antagonist WAY100635 (10 nM) followed by exposure to 6-OHDA (100 µM). Data are presented as means ± SEM (n = 3–6) and expressed as a percentage of the control group. **p < 0.01, and ***p < 0.001 vs. each control group. ^#p < 0.05 and ^###p < 0.001 between the two indicated groups.

Figure. 5

Mir-NCM promotes astrocyte proliferation via astrocytic 5-HT1A receptors. (a) Changes in the number of astrocytes after direct treatment with mirtazapine (2.5–10 µM) for 24 h. (b) Changes in the number of astrocytes after Mir (2.5–10 µM)-NCM treatment for 24 h. (c) Effect of WAY100635 (WAY) on the Mir-NCM-induced increase in the number of astrocytes. Astrocytes were treated with Mir-NCM (10 µM) and WAY (10 nM) for 24 h. Data are presented as means ± SEM (n = 20–24) and expressed as a percentage of the control group. *p < 0.05 and **p < 0.01 versus control-NCM treated group.

Figure. 6

Mir-NCM upregulated MT-1/2 expression in astrocytes via 5-HT1A receptors. (a,d,g) Representative microphotographs of GFAP (green), MT-1/2 (red), and Hoechst (blue) staining in astrocytes treated with mirtazapine (2.5–10 µM) (a), Mir (2.5–10 µM)-NCM (d), or Mir (5 μM)-NCM + WAY100635 (WAY; 10 nM) (g). Scale bar = 50 µM. (b,e,h) The ratio of MT-positive cells to all cells (c,f,i) Quantification of the optical density of MT immunoreactivity. Data are presented as means ± SEM (b,c: n = 18, e,f: n = 11–16, h,i: n = 25–30). *p < 0.05, **p < 0.01 and ***p < 0.001 versus control-NCM treated group. ^#p < 0.05 between the two indicated groups.

Figure. 7

Mirtazapine protects dopaminergic neurons by modulating MT-1/2 secretion from Mir-NCM-treated astrocytes via 5-HT1A receptors. (a) Neuroprotective effects of mirtazapine on astrocytes. Astrocytes were treated with Mir-NCM containing fresh mirtazapine (5 µM) with or without WAY100635 (WAY; 10 nM) for 24 h. Mesencephalic neurons were treated with Mir-NCM-ACM for 24 h and then exposed to 6-OHDA (50 µM). (b) Concentration of MT-1 in the conditioned medium from control-NCM or Mir-NCM-treated astrocytes. Astrocytes were treated with another flesh mirtazapine-added Mir-NCM for 24 h. Data are means ± SEM (n = 6). (c) MT-1/2 secreted from Mir-NCM-treated astrocytes protects dopaminergic neurons against 6-OHDA-induced toxicity. To neutralize MT-1/2 in Mir-NCM-ACM, Mir-NCM-ACM was preincubated with an anti-MT-1/2 antibody for 1 h (Mir-NCM-ACM + MT-1/2 Ab), and then applied to neuronal cultures. After treatment with Mir-NCM-ACM or Mir-NCM-ACM + MT-1/2 Ab for 24 h, neuronal cultures were treated with 6-OHDA (50 µM). Data are presented as means ± SEM (n = 6) and expressed as a percentage of the control group. **p < 0.01 and ***p < 0.001 versus each control group. ^#p < 0.05, ^##p < 0.01 between the two indicated groups.

Figure. 8

Schematic illustration of dopaminergic neuroprotective mechanisms of mirtazapine. Mirtazapine promotes the 5-HT release from serotonergic neuron by blocking adrenergic α2 receptors. Mirtazapine inhibits 5-HT2 and 5-HT3 receptor on astrocytes, which leads to selective 5-HT1A receptor stimulation and MT induction in astrocytes. MTs secreted from astrocytes attenuate oxidative stress and consequently protect dopaminergic neurons.