The high-affinity D2/D3 agonist D512 protects PC12 cells from 6-OHDA-induced apoptotic cell death and rescues dopaminergic neurons in the MPTP mouse model of Parkinson's disease

Abstract

In this study, in vitro and in vivo experiments were carried out with the high-affinity multifunctional D2/D3 agonist D-512 to explore its potential neuroprotective effects in models of Parkinson's disease and the potential mechanism(s) underlying such properties. Pre-treatment with D-512 in vitro was found to rescue rat adrenal Pheochromocytoma PC12 cells from toxicity induced by 6-hydroxydopamine administration in a dose-dependent manner. Neuroprotection was found to coincide with reductions in intracellular reactive oxygen species, lipid peroxidation, and DNA damage. In vivo, pre-treatment with 0.5 mg/kg D-512 was protective against neurodegenerative phenotypes associated with systemic administration of MPTP, including losses in striatal dopamine, reductions in numbers of DAergic neurons in the substantia nigra (SN), and locomotor dysfunction. These observations strongly suggest that the multifunctional drug D-512 may constitute a novel viable therapy for Parkinson's disease.

Keywords: 6-hydroxydopamine; MPTP; Parkinson's disease; multifunctional dopamine agonist; neuroprotection; oxidative stress.

Figure 1

Molecular structure of D-512

Figure 2

Dose-dependent effect of pretreatment as well as pretreatment followed by co-treatment of D-512 on cell viability of PC12 cells from toxicity induced by 75 μM 6-OHDA. (a) PC12 cells were treated with different concentrations of 6-OHDA (25 μM to 100 μM). (b) Dose-dependent effect of D-512 on cell viability. (c) PC12 cells were pretreated with different dosages of D-512 for 24 h followed by treatment with 75 μM 6-OHDA for 24 h. (d) PC12 cells were pretreated with different doses of D-512 for 24 h followed by co-treatment with 75 μM 6-OHDA for 24 h. Values shown are means ± SDs of three independent experiments performed in 4–6 replicates. One way ANOVA analysis (F (4,30) = 313.6, p < 0.0001 for 2a; F (8, 49) = 16.04, p < 0.0001) for 2c; F (8, 63) = 22.19, p < 0.0001 for 2d), followed by Tukey’s Multiple Comparison post hoc test were performed. **p<0.001 compared to the 6-OHDA or control groups; ##p<0.001 compared to the control group.

Figure 3

Effect of pretreatment with different concentrations of D-512 on ROS generation following treatment with 100 μM 6-OHDA in PC12 cells. (a) Dose-dependent effects of D-512 alone. (b) Cells were pretreated with D-512 for 24 h followed by co-treatment with 6-OHDA for another 24 h. Cells were then treated with 10 μM DCF-DA for 30 min and fluorescence intensity measured. Control data represents cells not treated with 6-OHDA and is represented as 100% with respect to the other groups. The values shown are means ± SDs of three independent experiments performed in triplicate or quadruple. One way ANOVA analysis (F (4,10) = 25.50, p < 0.0001) followed by Bonferroni’s Multiple Comparison post hoc test were performed. ##p<0.001 compared to the control group; **p<0.001 compared to the 6-OHDA + drug group.

Figure 4

Effect of pretreatment with different concentrations of D-512 on production of thiobarbituric acid reactive substances production induced by SNP (200 μM). Control data represents cells not treated with SNP and is represented as 100% with respect to the other groups. The values shown are means ± SDs of three independent experiments performed in triplicate or quadruple. One way ANOVA analysis (F (5, 10) = 7.984, p < 0.0029) followed by Bonferroni’s Multiple Comparison post hoc test were performed. ##p<0.01 compared to the control group; **p<0.05 compared to the SNP + drug group.

Figure 5

(a) Effect of D-512 on 6-OHDA-induced nucleic condensation. Apoptotic nuclei were visualized via fluorescence dye 33342 staining. Apoptotic cells with high fluorescence intensity are indicated by arrows. Scale bar: 200 μm. (b) Effect of pretreatment with varying concentration of D-512 followed by co-treatment with 75 μM 6-OHDA on DNA fragmentation in PC12 cells. Lanes 1–4: marker, DNA laddering of control cells, DNA laddering in response to 75 μM 6-OHDA alone, or 10 μM D-512 alone. Lanes 5–7: DNA laddering in response to pretreatment with varying concentrations of D-512 (10 μM, 5 μM and 1 μM) along with co-treatment with 75 μM 6-OHDA + 10 μM D-512, 75 μM 6-OHDA + 5 μM D-512, or 75 μM 6-OHDA + 1 μM D-512.

Figure 5

(a) Effect of D-512 on 6-OHDA-induced nucleic condensation. Apoptotic nuclei were visualized via fluorescence dye 33342 staining. Apoptotic cells with high fluorescence intensity are indicated by arrows. Scale bar: 200 μm. (b) Effect of pretreatment with varying concentration of D-512 followed by co-treatment with 75 μM 6-OHDA on DNA fragmentation in PC12 cells. Lanes 1–4: marker, DNA laddering of control cells, DNA laddering in response to 75 μM 6-OHDA alone, or 10 μM D-512 alone. Lanes 5–7: DNA laddering in response to pretreatment with varying concentrations of D-512 (10 μM, 5 μM and 1 μM) along with co-treatment with 75 μM 6-OHDA + 10 μM D-512, 75 μM 6-OHDA + 5 μM D-512, or 75 μM 6-OHDA + 1 μM D-512.

Figure 6

Effect of D512 on motor coordination and balance in the pole test. One way ANOVA analysis (F (3, 12) = 58.22, p < 0.0001) indicates *p<0.001 between vehicle/sal and veh/MPTP; # p<0.001 between veh/MPTP and D512/MPTP (n=4).

Figure 7

a.) Effect of pretreatment of 0.5 mg/kg D512 for three days followed by two days co-treatment with MPTP (20 mg/kg, 12 h apart) on striatal DA levels. One way ANOVA analysis (F (3, 9) = 74.59, p < 0.0001) ** p < 0.001 between vehicle/Sal and Vehicle/MPTP; # p<0.01 between D512/MPTP and Vehicle/MPTP (n=4). (b) MPP⁺ levels were measured in striatal tissues collected from C57BL 6 mice treated with vehicle MPTP or D512 MPTP. Tissue was harvested 90 min post-MPTP injection (20 mg/kg) and analyzed via HPLC with electrochemical detection. Values are presented as ng MPP⁺/mg protein, n = 4 per treatment condition.

Figure 8

(a) Stereological quantification of TH-positive DAergic cell counts within the SNpc. One way ANOVA analysis (F (3, 24) = 117.2, p <0.0001) indicates ** p < 0.001 between Vehicle/Sal and Vehicle/MPTP; ## p < 0.001 between D512/MPTP and Vehicle/MPTP D512 at 0.5mg/Kg concentration (n=8). (b) Representative photomicrographs of the SN with TH immunohistochemistry (10X).

Figure 8

(a) Stereological quantification of TH-positive DAergic cell counts within the SNpc. One way ANOVA analysis (F (3, 24) = 117.2, p <0.0001) indicates ** p < 0.001 between Vehicle/Sal and Vehicle/MPTP; ## p < 0.001 between D512/MPTP and Vehicle/MPTP D512 at 0.5mg/Kg concentration (n=8). (b) Representative photomicrographs of the SN with TH immunohistochemistry (10X).