Targeting alpha synuclein and amyloid beta by a multifunctional, brain-penetrant dopamine D2/D3 agonist D-520: Potential therapeutic application in Parkinson's disease with dementia

Abstract

A significant number of people with Parkinson's disease (PD) develop dementia in addition to cognitive dysfunction and are diagnosed as PD with dementia (PDD). This is characterized by cortical and limbic alpha synuclein (α-syn) accumulation, and high levels of diffuse amyloid beta (Aβ) plaques in the striatum and neocortical areas. In this regard, we evaluated the effect of a brain-penetrant, novel multifunctional dopamine D2/D3 agonist, D-520 on the inhibition of Aβ aggregation and disintegration of α-syn and Aβ aggregates in vitro using purified proteins and in a cell culture model that produces intracellular Aβ-induced toxicity. We further evaluated the effect of D-520 in a Drosophila model of Aβ_1-42 toxicity. We report that D-520 inhibits the formation of Aβ aggregates in vitro and promotes the disaggregation of both α-syn and Aβ aggregates. Finally, in an in vivo Drosophila model of Aβ_1-42 dependent toxicity, D-520 exhibited efficacy by rescuing fly eyes from retinal degeneration caused by Aβ toxicity. Our data indicate the potential therapeutic applicability of D-520 in addressing motor dysfunction and neuroprotection in PD and PDD, as well as attenuating dementia in people with PDD.

Figure 1

Mode of action for multifunctional activity of D-520.

Figure 2

Effect of D-520 on disaggregation of α-synuclein aggregates formed by seeding: (A) Aggregates formed by incubating 1.25 mg/mL α-syn with 0.5% PFFs for a period of 30D without shaking were incubated with D-520 for a period of 15 days. The ability of D-520 to dissociate the aggregates was studied by ThT assay at 10D and 15D of incubation. Values are represented in terms of % 0D aggregated synuclein which represents the aggregates collected at 30D from seeding. (B) Viability of PC12 cells was measured by MTT assay after 24 h treatment with aggregated synuclein incubated with D-520 collected at 10D and 15D. Values were normalized to control. Data values shown are means ± SD of three independent experiments. One-way ANOVA analysis followed by Tukey’s multiple comparison post hoc test was performed, ^****p < 0.0001 and ^**p < 0.01 compared to aggregated Syn-0D,; ^$$$$p ≤ 0.0001 compared to aggregated synuclein-10D and ^####p ≤ 0.0001 compared to aggregated synuclein-15D.

Figure 3

TEM analysis of 30D 0.5% PFF seeded α-syn aggregates. (A) Day 0, (B) Day 15, (C) Day 15 in the presence of D-520; Scale bar = 100 nm.

Figure 4

Kinetics on inhibition and dissociation of Aβ_1–42 oligomer formation by D-520: (A–C) ThT fluorescence of Aβ_1–42 (10 μM) with or without incubation with either D-520 (20 μM, Panel: A) or D-520 (10 μM, Panel: B) or D-520 (1 or, 0.1 or 0.01 μM, Panel: C) for 24 h. (D) ThT fluorescence of Aβ_1–42 fibrils (10 μM) with or without incubation with D-520 (20 μM) for 24 h. The ThT fluorescence of Aβ_1–42 at 0 h was considered as 100%. One-way ANOVA followed by Tukey’s multiple comparison post hoc test, ****P < 0.0001 comparing abeta to abeta + D520 (Panel A-C) and ****P < 0.0001 comparing abeta fibrils to abeta fibril + D520 (Panel D).

Figure 5

D-520 protects the cells from toxicity caused by Aβ oligomers: MC65 cells were plated in a 96 well plate. Cells received either complete media, or opti MEM with or without Tetracycline. The cells which received media without Tetracycline were treated with different concentrations of D-520 (1–30 μM) for a period of 48 h. Viability was measured by MTT assay. The values were plotted taking control as 100%. Data values shown are means ± SD of three independent experiments. One-way ANOVA analysis followed by Tukey’s multiple comparison post hoc test was performed, *p ≤ 0.1 and ****p ≤ 0.0001 compared to controls grown in complete media with Tetracycline.

Figure 6

D-520 diminishes the intracellular population of Aβ oligomers: Western blots of extracts from MC65 cell lines that conditionally expresses C99, a 99-residue carboxyl terminal fragment of APP in the absence of tetracycline. C99 is subsequently cleaved by cellular γ-secretase to generate Aβ. The Aβ oligomers are seen when tetracycline is removed from the media. D-520 blocks the formation of Aβ oligomers. Immuno blotting was carried out using the antibody NAB-228 from Cell signalling (upper panel, Panel A)) and GAPDH was used as the loading control. The immuno blot images were quantified in the lower panel (Panel B). The plot shown is representative of 3 replicates. Standard marker as shown in the figure is part of the same gel (see the supplementary material section) and was developed with fluorescence exposure as described in the method section. The results shown is representative of 3 replicates, **p < 0.01, ***p < 0.001 compared to –Tet and $$$p < 0.001 compared to –Tet.

Figure 7

D-520 blocks the appearance of Aβ oligomers in MC65 cells: Immunocytochemistry of MC65 cells from treatment with D-520: MC65 cells were treated with the D-520 at a concentration of 20 μM immediately after the removal of Tetracycline from the media. 48 h after the treatments, cells were fixed and immune fluorescently labeled with Aβ specific antibody (Green) and counter stained with DAPI for nuclear staining. Panel A: control shows the dispersed expression of Aβ. When Tetracycline is removed from the media, it leads to the formation of Aβ oligomers seen as punctate structures with intense green staining (Panel B). However, when the cells are treated with D-520, it leads to the decrease in the Aβ oligomer formation as seen by the even fluorescence throughout the cells (Panel C).

Figure 8

(A) Representative images of fly eyes expressing, or not, Aβ_1–42 in fly eyes, driven by the GMR-Gal4 driver. Flies were 14 days old and were either fed with the vehicle control (ultrapure water) or D-520 (1 mg/mL dissolved in ultrapure water) for 14 days, at which time heads were dissected, imaged and quantified, as summarized in (B). Control: GMR-Gal4 driver and UAS-CD8-GFP in the absence of Aβ_1–42. (B) Shown in histograms are GFP fluorescence means −/+ standard deviation. Statistical analyses were conducted with ANOVA. ^***P < 0.001 comparing columns 1, 2 and 4 to column 3.