Zn homeostasis in genetic models of Parkinson's disease in Caenorhabditis elegans

Abstract

While the underlying mechanisms of Parkinson's disease (PD) are still insufficiently studied, a complex interaction between genetic and environmental factors is emphasized. Nevertheless, the role of the essential trace element zinc (Zn) in this regard remains controversial. In this study we altered Zn balance within PD models of the versatile model organism Caenorhabditis elegans (C. elegans) in order to examine whether a genetic predisposition in selected genes with relevance for PD affects Zn homeostasis. Protein-bound and labile Zn species act in various areas, such as enzymatic catalysis, protein stabilization pathways and cell signaling. Therefore, total Zn and labile Zn were quantitatively determined in living nematodes as individual biomarkers of Zn uptake and bioavailability with inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) or a multi-well method using the fluorescent probe ZinPyr-1. Young and middle-aged deletion mutants of catp-6 and pdr-1, which are orthologues of mammalian ATP13A2 (PARK9) and parkin (PARK2), showed altered Zn homeostasis following Zn exposure compared to wildtype worms. Furthermore, age-specific differences in Zn uptake were observed in wildtype worms for total as well as labile Zn species. These data emphasize the importance of differentiation between Zn species as meaningful biomarkers of Zn uptake as well as the need for further studies investigating the role of dysregulated Zn homeostasis in the etiology of PD.

Keywords: Caenorhabditis elegans; Labile zinc; Parkinson disease; Zinc; Zinc homeostasis.

Figure 1:

Survival of WT, catp-6Δ and pdr-1Δ after exposure to 200 μM TPEN, 200 μM or 500 μM Zn. Survival rates of [A]: day 2 adults and [B]: day 5 adults evaluated after 24 h of feeding with enriched OP50 E. coli of the respective C. elegans strains. Data are expressed as means of 4 independent determinations + SEM. *: p < 0.05, **: p < 0.01, and ***: p < 0.001 compared to WT controls.

Figure 2:

Total Zn in WT, catp-6Δ and pdr-1Δ following TPEN or Zn exposure. Zn concentrations in [A]: day 2 adults and [B]: day 5 adults of the respective C. elegans strains were determined using ICP-MS/MS after exposing L4 larvae or day 4 adults to 200 μM TPEN, 200 μM or 500 μM Zn via feeding enriched OP50 E. coli for 24 h. Data are expressed as means of at least 4 determinations + SEM normalized to worms’ weight. *: p < 0.05, **: p < 0.01, and ***: p < 0.001 compared to WT controls. §: p < 0.05, §§: p < 0.01, and §§§: p < 0.001 compared to respective same conditions in day 2 adults.

Figure 3:

[Zn]²⁺ visualization after loading of C. elegans with ZinPyr-1. Fluorescence images of ZinPyr-1 signal in living WT day 2 adults after exposure to 200 μM TPEN, 200 μM and 500 μM Zn via feeding enriched OP50 E. coli for 24 h and respective bright field images. Scale bars represent 100 μm.

Figure 4:

[Zn]²⁺ in WT, catp-6Δ and pdr-1Δ following TPEN or Zn exposure. [Zn]²⁺ in [A]: day 2 adults and [B]: day 5 adults of the respective C. elegans strains after exposing L4 larvae or day 4 adults to 200 μM TPEN, 200 μM or 500 μM Zn via feeding enriched OP50 E. coli for 24 h. Determination was realized by measuring [Zn]²⁺-dependent fluorescence as the difference between F_max and F_min as previously described [26]. Data are expressed as means of at least 3 determinations + SEM normalized to WT control. *: p < 0.05, **: p < 0.01, and ***: p < 0.001 compared to WT controls. §: p < 0.05, §§: p < 0.01, and §§§: p < 0.001 compared to respective same conditions in day 2 adults.