2,4-Dichlorophenoxyacetic Acid Induces Degeneration of mDA Neurons In Vitro

Abstract

Background: Parkinson's disease (PD) affects 1-2% of the population over the age of 60 and the majority of PD cases are sporadic, without any family history of the disease. Neuroinflammation driven by microglia has been shown to promote the progression of midbrain dopaminergic (mDA) neuron loss through the release of neurotoxic factors. Interestingly, the risk of developing PD is significantly higher in distinct occupations, such as farming and agriculture, and is linked to the use of pesticides and herbicides. Methods: The neurotoxic features of 2,4-Dichlorophenoxyacetic acid (2,4D) at concentrations of 10 µM and 1 mM were analyzed in two distinct E14 midbrain neuron culture systems and in primary microglia. Results: The application of 1 mM 2,4D resulted in mDA neuron loss in neuron-enriched cultures. Notably, 2,4D-induced neurotoxicity significantly increased in the presence of microglia in neuron-glia cultures, suggesting that microglia-mediated neurotoxicity could be one mechanism for progressive neuron loss in this in vitro setup. However, 2,4D alone was unable to trigger microglia reactivity. Conclusions: Taken together, we demonstrate that 2,4D is neurotoxic for mDA neurons and that the presence of glia cells enhances 2,4D-induced neuron death. These data support the role of 2,4D as a risk factor for the development and progression of PD and further suggest the involvement of microglia during 2,4D-induced mDA neuron loss.

Keywords: 2,4-Dichlorophenoxyacetic acid; TNFα; mDA neurons; microglia; neurodegeneration.

Figure 1

2,4D treatment results in reduced number of TH⁺ neurons in E14 midbrain neuron-enriched cultures. Scheme depicting experimental workflow (A). Created using BioRender.com. Neuron-enriched cultures were left untreated as control cells (B) or were treated with ethanol (EtOH, (C)), 2,4D 1 mM (D) and 2,4D 10 µM (E) for 48 h. Representative images of TH⁺ neurons for each treatment group are depicted. Scale bar indicates 100 µm. Quantification of TH⁺ neurons in different treatment groups (F). Levels of TNFα in supernatants from neuron-enriched cultures after indicated treatments (G). Data are given as means ± SEM from at least three independent experiments. p-values derived from one-way ANOVA followed by Tukey´s multiple comparisons tests are * p < 0.05 and ** p < 0.01.

Figure 2

Enhanced loss of TH⁺ neurons in E14 midbrain neuron-glia cultures after 2,4D treatment. Scheme depicting experimental workflow (A). Created using BioRender.com. Neuron-glia cultures were left untreated (B) or were treated with ethanol (EtOH, (C)), 2,4D 1mM (D) and 2,4D 10 µM (E) for 48 h under serum-free conditions. Representative images of labelled TH⁺ neurons for each treatment group are shown. Scale bar indicates 100 µm. Quantification of TH⁺ neurons in different treatment groups (F). TNFα levels in supernatants after indicated treatments (G). Data are given as means ± SEM from at least three independent experiments. p-values derived from one-way ANOVA followed by Tukey´s multiple comparisons tests are ** p < 0.01 and **** p < 0.0001.

Figure 3

2,4D treatment of microglia cultures is not sufficient to trigger primary microglia reactivity. Scheme showing experimental workflow for primary microglia cultures (A). Created using BioRender.com. Microglia cultures were left untreated or were treated with IFNγ 10 ng/mL or 2,4D 1 mM, and microglia morphology after IB4-labelling (green) is depicted as a proxy for microglia reactivity (B). Scale bar indicates 50 µm. Treatment of microglia with IFNγ at 1 ng/mL or 10 ng/mL for 24 h under serum-free conditions resulted in increased microglia numbers (C) and increased release of TNFα (D) and IL6 (E). No changes in numbers of viable microglia (F) and microglial TNFα (G) and IL6 (H) secretion could be observed after treatment with 2,4D for 24 h. Data are given as means ± SEM from at least three independent experiments. p-values derived from one-way ANOVA followed by Tukey´s multiple comparisons tests are * p < 0.05 and ** p < 0.01.