Role of Caenorhabditis elegans AKT-1/2 and SGK-1 in Manganese Toxicity

Abstract

Excessive levels of the essential metal manganese (Mn) may cause a syndrome similar to Parkinson's disease. The model organism Caenorhabditis elegans mimics some of Mn effects in mammals, including dopaminergic neurodegeneration, oxidative stress, and increased levels of AKT. The evolutionarily conserved insulin/insulin-like growth factor-1 signaling pathway (IIS) modulates worm longevity, metabolism, and antioxidant responses by antagonizing the transcription factors DAF-16/FOXO and SKN-1/Nrf-2. AKT-1, AKT-2, and SGK-1 act upstream of these transcription factors. To study the role of these proteins in C. elegans response to Mn intoxication, wild-type N2 and loss-of-function mutants were exposed to Mn (2.5 to 100 mM) for 1 h at the L1 larval stage. Strains with loss-of-function in akt-1, akt-2, and sgk-1 had higher resistance to Mn compared to N2 in the survival test. All strains tested accumulated Mn similarly, as shown by ICP-MS. DAF-16 nuclear translocation was observed by fluorescence microscopy in WT and loss-of-function strains exposed to Mn. qRT-PCR data indicate increased expression of γ-glutamyl cysteine synthetase (GCS-1) antioxidant enzyme in akt-1 mutants. The expression of sod-3 (superoxide dismutase homologue) was increased in the akt-1 mutant worms, independent of Mn treatment. However, dopaminergic neurons degenerated even in the more resistant strains. Dopaminergic function was evaluated with the basal slowing response behavioral test and dopaminergic neuron integrity was evaluated using worms expressing green fluorescent protein (GFP) under the dopamine transporter (DAT-1) promoter. These results suggest that AKT-1/2 and SGK-1 play a role in C. elegans response to Mn intoxication. However, tissue-specific responses may occur in dopaminergic neurons, contributing to degeneration.

Keywords: Akt/PKB; C. elegans; DAF-16; Manganese; SGK-1; Signaling pathways.

Fig. 1

C. elegans (L1) survival after exposure to Mn (2.5–100 mM) for 1 h. Controls were incubated with 85 mM NaCl. The number of surviving worms was determined 48 h after treatment in N2 (wild-type, WT) or null mutants akt-1(ok525), akt-2(ok593), sgk-1(ok538) or gain-of-function mutant akt-1(mg144). (A) Data represent the percentage of surviving worms relative to day 0 and are expressed as mean ± S.E.M. of 3 to 6 experiments and is plotted against the logarithmic scale of Mn concentrations. (B) Lethal dose 50% (LD50) was calculated by non-linear regression. ** P <0.01 *** p <0.001 compared with WT group, two-way ANOVA followed by post hoc Tukey’s test.

Fig. 2

Intraworm manganese (A – B) or calcium (C – D) content after 1 h treatment with 10 or 50 mM MnCl₂ was quantified by ICP-MS immediately after treatment (0 h) or 48 h after. Metal content is expressed as μg/mg protein. Data are expressed as mean ± S.E.M. from three independent experiments. Statistical analysis by two-way ANOVA followed by post hoc Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001 compared to same strain control.

Fig. 3

Mn induces DAF-16 nuclear translocation. Akt-1(ok525), akt-2(ok593), sgk-1(ok538) were crossed with Pdaf-16A::GFP. Worms were exposed to 10 or 50 mM MnCl₂ for 1 h. Controls were incubated with 85 mM NaCl. Worms were scored as having cytoplasmic (A), intermediate (B) or nuclear (C) DAF-16 distribution. DAF-16 was visualized by fluorescent microscopy within 10 min of Mn exposure (D), or at the end of 1 h exposure (E). Results are expressed as percent of worms with each condition. Data are expressed as mean + S.E.M. from five independent experiments. Statistical analysis by two-way ANOVA followed by post hoc Tukey’s test. Representative images were obtained with a PerkinElmer spinning disk confocal, 40X objective. Scale bar represents 10μm.

Fig. 4

N2 (WT) or null mutants akt-1(ok525), akt-2(ok593), sgk-1(ok538) or gain-of-function mutant akt-1(mg144) were exposed to Mn for 1 h (10 or 50 mM). Controls were incubated with 85 mM NaCl. The worms were homogenized after treatment. Expression of sod-3 (A), gst-4 (B), skn-1 (C) and gcs-1 (D) relative to the constitutive gene afd-1 (β-actin) was normalized to the N2 control group. Results are expressed as mean ± S.E.M. of 3 to 4 experiments. (E) GSH levels were determined using a standard curve (μM GSH/mg protein). Results are expressed as percentage GSH content relative to same strain control and mean ± S.E.M. of 3 experiments. * p <0.05, ** p <0.01, *** p <0.001 compared to the N2 control group. # p <0.05, ## p <0.01 compared to group 50 mM N2. Two-way ANOVA followed by post hoc Tukey’s test.

Fig. 5

Morphology of CEPs and ADEs neurons DAergic neurons. Worms were evaluated 2 h after an acute treatment with MnCl₂ (10 or 50 mM) by fluorescence microscopy (X 40 magnification). Controls were incubated with 85 mM NaCl. Worms with healthy neurons were scored as normal (A). Worms with any of the following changes in their dopaminergic neurons were quantified as containing degeneration: (B) puncta (discontinuous marking the dendrite, arrows), (C) loss or cell body shrinkage (arrowhead), loss of dendrites (asterisks), exemplified in the confocal images. (D) Data represent the percentage of worms with degeneration. Results are expressed as mean ± S.E.M. of 6 experiments. * P <0.05, *** p <0.001 compared to the control group. Two-way ANOVA followed by post hoc Tukey’s test. Representative images were obtained with a PerkinElmer spinning disk confocal, 40X objective. Scale bar represents 10 μm.

Fig. 6

Basal slowing response was evaluated in WT N2 and null mutants akt-1(ok525), akt-2(ok593), sgk-1(ok538) and gain of function mutant akt-1(mg144) exposed to Mn for 1 h (10 or 50 mM). Controls were incubated with 85 mM NaCl. The test was performed 48 h after Mn exposure. Worms were washed off plates with S-basal and 5 worms were pipetted in plates with or without OP-50 E.coli (food). Worms were allowed to acclimate to the new plates for 5 min and then body bends were counted in 20 s intervals for each worm to obtain an average. Basal slowing response is expressed as number of body bends off food minus the number of body bends on food (Δ). Cat-2 mutants, with deficiency in tyrosine hydroxylase, were used as positive control. Data are expressed as mean ± S.E.M. of 3 to 5 experiments. * P <0.05, ** p <0.01, compared to the N2 control group. Two-way ANOVA followed by post hoc Tukey’s test.

Fig. 7

Phosphorylated proteins were evaluated by western blotting. Worms were exposed to Mn for 1 h (10 or 50 mM). Samples for SDS-PAGE were prepared immediately after treatment. Blots were developed by chemiluminescence. Bands were quantified by densitometry with imageJ software. (A) Phosphorylation of JNK in wild-type N2 or null mutants (B) akt-1(ok525), (C) akt-2(ok593), (D) sgk-1(ok538). Phosphorylated (P) JNK was normalized to total (T) JNK. (E) Phosphorylation of AAK/AMPK in wild-type N2 or null mutants (F) akt-1(ok525), (G) akt-2(ok593), (H) sgk-1(ok538). P AMPK was normalized to β-actin. Data represent fold change compared to controls and express the mean ± S.E.M. of 4 experiments. ** p < 0.01, *** p < 0.001 compared to controls (one-way ANOVA followed by post hoc Tukey’s test).

Figure 8

Schematic representation of our findings. Previous work by our group reported increased AKT levels in worms exposed to Mn (Avila et al., 2012). Upstream of AKT-1/2 and SGK-1 is DAF-2 (the only receptor similar to mammalian insulin-like growth factor 1, IGF-1, receptor), activated by insulin-like peptides. DAF-2 activates AGE-1 (homologous of phosphatidyl inositol-3 kinase, PI3K), which converts phosphatidyl inositol-2 phosphate (PIP2) to PIP3 and activates phosphatidyl inositol-dependent kinase (PDK-1). PDK-1 activates AKT-1/2 and SGK-1, which in turn phosphorylate SKN-1 and DAF-16 at various sites, retaining the transcription factors in the cytoplasm. Our data indicate that AKT participates in Mn toxicity by preventing DAF-16 translocation to the nucleus. In AKT deficient mutants, we see increased DAF-16 nuclear localization (Fig. 3) and increased gcs-1 and sod-3 mRNA, which are DAF-16 targets (Fig. 4). This antioxidant response may be responsible for increased resistance to Mn exposure (Fig. 1) in mutant strains. This resistance is not observed in worm dopaminergic neurons. SGK-1 levels as well as SKN-1 nuclear translocation remain to be tested.