Prolongation of metallothionein induction combats Aß and α-synuclein toxicity in aged transgenic Caenorhabditis elegans

Abstract

Neurodegenerative disorders (ND) like Alzheimer's (AD), Parkinson's (PD), Huntington's or Prion diseases share similar pathological features. They are all age dependent and are often associated with disruptions in analogous metabolic processes such as protein aggregation and oxidative stress, both of which involve metal ions like copper, manganese and iron. Bush and Tanzi proposed 2008 in the 'metal hypothesis of Alzheimer's disease' that a breakdown in metal homeostasis is the main cause of NDs, and drugs restoring metal homeostasis are promising novel therapeutic strategies. We report here that metallothionein (MT), an endogenous metal detoxifying protein, is increased in young amyloid ß (Aß) expressing Caenorhabditis elegans, whereas it is not in wild type strains. Further MT induction collapsed in 8 days old transgenic worms, indicating the age dependency of disease outbreak, and sharing intriguing parallels to diminished MT levels in human brains of AD. A medium throughput screening assay method was established to search for compounds increasing the MT level. Compounds known to induce MT release like progesterone, ZnSO₄, quercetin, dexamethasone and apomorphine were active in models of AD and PD. Thioflavin T, clioquinol and emodin are promising leads in AD and PD research, whose mode of action has not been fully established yet. In this study, we could show that the reduction of Aß and α-synuclein toxicity in transgenic C. elegans models correlated with the prolongation of MT induction time and that knockdown of MT with RNA interference resulted in a loss of bioactivity.

Figure 1

Preliminary tests for a MT medium throughput screening assay based on transgenic C. elegans. (a) Fluorescence of MT::gfp in strains CL2122 and CL2120 was measured by a fluorescence multiwell plate reader at em/ex 450/535. (b) Fluorescence in strains CL2120 and CL2122 was detected at day 6 by fluorescent microscope (magnification ×20). (c) Serial dilution (1:1) of CL2120 L4 larvae in a 96 well plate starting with 512 worms/well in triplicates. Fluorescence of reporter was measured after 3, 6 and 9 days. (d) Serial dilution (1:1) of Escherichia coli strain OP50 in a 96 well plate starting with a concentration of 6 mg/ml. Absorbance was detected by a multiwellplate reader at 600 nm. OD₆₀₀ = 0,8 at a concentration of 5 mg/ml. (e) Influence of different DMSO concentrations on fluorescence of GFP reporter in strains CL2120, CL2659 and NL5901. *p ≤ 0.05; **p ≤ 0.005.

Figure 2

Influence of different concentrations of MT inducing test compounds on strains CL2120, CL2659, NL5901 and N2 was measured by fluorescence multiwell plate reader at em/ex 450/535. Each concentration was tested in triplicates. Change in fluorescence of test compounds was compared to vehicle control 1% DMSO in all assays. (a) ZnSO₄ (b) apomorphin (APM) (c) dexamethasone (DXM) (d) quercetin. Error bars show s.d. *p ≤ 0.05; **p ≤ 0.005; QC quercetin, LD levodopa, d day.

Figure 3

Influence of different concentrations of neuroprotective test compounds on strains CL2120, CL2659, NL5901 and N2 was measured by fluorescence multiwell plate reader at em/ex 450/535. Each concentration was tested in triplicates. Change in fluorescence of test compounds was compared to vehicle control 1% DMSO in all assays. (a) thioflavin T (Th T) (b) clioquinol (CQL) (c) emodin (d) sesamin. Error bars show s.d. *p ≤ 0.05; **p ≤ 0.005; QC quercetin, LD levodopa, d day.

Figure 4

Relative expression of mt-1 (a) and mt-2 (b) following treatment with test compounds. 150 worms of CL2120 strain were treated with 40 µM emodin, 100 µg/ml clioquinol and 100 µM ZnSO₄. Relative fold change was measured using comparative ΔΔCT method. Gene expression data was normalized to rps-18. (c) 150 worms of CL2120 strain were treated with 40 µM emodin for a period of 3 and 5 days and quantitative RT PCR was performed to measure relative mt-1 and mt-2 expression. One-way ANOVA followed by Tukey's multiple comparisons test was used to measure statistical significance (*p ≤ 0.01). All experiments were performed in triplicates. Data represents means ± SEM.