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.
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Funding
TVP received a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Foundation, Ministry of Education of Brazil, (Proc. #0407/13-5). LA received a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 202662/2014-4 - SWE). MA and ABB were supported by National Institute of Healt (NIH) R01 ES10563. MA was also supported by R01 ES07331 and R01 ES020852. We thank the “Deutsche Forschungsgemeinschaft” (DFG) further for the financial support of Schw 903/9-1 and BO 4103/2-1. Images were obtained at the Analytical Imaging Facility of the Albert Einstein College of Medicine [NCI cancer center support grant (P30CA013330)]. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Funding agencies had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Supplemental Fig.1
Confocal microscopy images depicting alterations in GFP marking of dopaminergic neurons. A and B depict a normal worm. C depicts a worm with puncta (arrow heads). D depicts a worm with a shrunken soma (arrows). C and D were considered worms with degeneration. Worms were in the L1 stage. Green scale bar = 10 μm (×40 objective). White scale bar = 13 μm (×60 objective). (PNG 3287 kb)
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Peres, T.V., Arantes, L.P., Miah, M.R. et al. Role of Caenorhabditis elegans AKT-1/2 and SGK-1 in Manganese Toxicity. Neurotox Res 34, 584–596 (2018). https://doi.org/10.1007/s12640-018-9915-1
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DOI: https://doi.org/10.1007/s12640-018-9915-1