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Quantitative proteomics analysis of zebrafish exposed to sub-lethal dosages of β-methyl-amino-L-alanine (BMAA).

Frøyset AK, Khan EA, Fladmark KE - Sci Rep (2016)

Bottom Line: The exposed larvae showed no developmental abnormalities, but a reduced heart rate and increased expression of GSK3 isoforms.Seven of these proteins could be associated to glutamate receptor signaling and recycling.We also found that BMAA influenced the endocannabinoid system by up-regulation of fatty acid amide hydrolase (FAAH) and that FAAH inhibitor URB597 reduced the BMAA effect on heart rate and GSK3 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Bergen, Thormøhlensgate 55, 5020 Bergen, Norway.

ABSTRACT
The non-protein amino acid β-methylamino-L-alanine (BMAA) is a neurotoxin present in microalgae and shown to accumulate in the food web. BMAA has been linked to the complex neurodegenerative disorder of Guam and to increased incidents sporadic ALS. Two main neurotoxic routes are suggested; an excitotoxic by acting as an agonist towards glutamate receptors and a metabolic by misincorporating into cellular proteins. We have used zebrafish, an increasingly used model for neurodegenerative diseases, to further identify signaling components involved in BMAA-induced toxicity. Zebrafish embryos were exposed to sub-lethal dosages of BMAA and a label-free proteomics analysis was conducted on larvae 4 days post fertilization. The exposed larvae showed no developmental abnormalities, but a reduced heart rate and increased expression of GSK3 isoforms. Search towards a reviewed database containing 2968 entries identified 480 proteins. Only 17 of these were regulated 2-fold or more in the exposed larvae. Seven of these proteins could be associated to glutamate receptor signaling and recycling. The remaining nine have all been linked to disturbance in protein homeostasis, reactive oxygen species (ROS) development or neuronal cell death. We also found that BMAA influenced the endocannabinoid system by up-regulation of fatty acid amide hydrolase (FAAH) and that FAAH inhibitor URB597 reduced the BMAA effect on heart rate and GSK3 expression.

No MeSH data available.


Related in: MedlinePlus

Embryonal BMAA-injection increases GSK3α/β expression in larvae.Embryos injected with 6.5 ng BMAA or solvent control at one-cell stage were harvested 4 dpf. Proteins were extracted from whole larvae (n > 50) and subjected to Western blotting using anti-GSK-3α/β. Ponceau-S staining was used as loading control. Blot shows control and BMAA-injected samples from two individual experiments.
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f2: Embryonal BMAA-injection increases GSK3α/β expression in larvae.Embryos injected with 6.5 ng BMAA or solvent control at one-cell stage were harvested 4 dpf. Proteins were extracted from whole larvae (n > 50) and subjected to Western blotting using anti-GSK-3α/β. Ponceau-S staining was used as loading control. Blot shows control and BMAA-injected samples from two individual experiments.

Mentions: Since the BMAA-injected larvae showed no signs of phenotypic abnormalities compared to what was observed by Purdie et al.27, we also determined whether the BMAA-injected larvae showed an induction of GSK3 isoforms. Previous studies in neuroblastoma cells and cerebellum of BMAA-exposed rats have shown that the levels of both α and β isoforms of GSK3 increase as a response to BMAA2829. We analyzed GSK3 α/β levels in total lysates of BMAA (6.5 ng) and control injected 4 dpf larvae using Western blotting. As can be seen in Fig. 2 BMAA-injected embryos showed increased levels of both α and β isoforms of GSK3 at 4 dpf. Larvae (4 dpf) injected with 6.5 ng BMAA were therefore selected for further label-free proteomics analysis.


Quantitative proteomics analysis of zebrafish exposed to sub-lethal dosages of β-methyl-amino-L-alanine (BMAA).

Frøyset AK, Khan EA, Fladmark KE - Sci Rep (2016)

Embryonal BMAA-injection increases GSK3α/β expression in larvae.Embryos injected with 6.5 ng BMAA or solvent control at one-cell stage were harvested 4 dpf. Proteins were extracted from whole larvae (n > 50) and subjected to Western blotting using anti-GSK-3α/β. Ponceau-S staining was used as loading control. Blot shows control and BMAA-injected samples from two individual experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940735&req=5

f2: Embryonal BMAA-injection increases GSK3α/β expression in larvae.Embryos injected with 6.5 ng BMAA or solvent control at one-cell stage were harvested 4 dpf. Proteins were extracted from whole larvae (n > 50) and subjected to Western blotting using anti-GSK-3α/β. Ponceau-S staining was used as loading control. Blot shows control and BMAA-injected samples from two individual experiments.
Mentions: Since the BMAA-injected larvae showed no signs of phenotypic abnormalities compared to what was observed by Purdie et al.27, we also determined whether the BMAA-injected larvae showed an induction of GSK3 isoforms. Previous studies in neuroblastoma cells and cerebellum of BMAA-exposed rats have shown that the levels of both α and β isoforms of GSK3 increase as a response to BMAA2829. We analyzed GSK3 α/β levels in total lysates of BMAA (6.5 ng) and control injected 4 dpf larvae using Western blotting. As can be seen in Fig. 2 BMAA-injected embryos showed increased levels of both α and β isoforms of GSK3 at 4 dpf. Larvae (4 dpf) injected with 6.5 ng BMAA were therefore selected for further label-free proteomics analysis.

Bottom Line: The exposed larvae showed no developmental abnormalities, but a reduced heart rate and increased expression of GSK3 isoforms.Seven of these proteins could be associated to glutamate receptor signaling and recycling.We also found that BMAA influenced the endocannabinoid system by up-regulation of fatty acid amide hydrolase (FAAH) and that FAAH inhibitor URB597 reduced the BMAA effect on heart rate and GSK3 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Bergen, Thormøhlensgate 55, 5020 Bergen, Norway.

ABSTRACT
The non-protein amino acid β-methylamino-L-alanine (BMAA) is a neurotoxin present in microalgae and shown to accumulate in the food web. BMAA has been linked to the complex neurodegenerative disorder of Guam and to increased incidents sporadic ALS. Two main neurotoxic routes are suggested; an excitotoxic by acting as an agonist towards glutamate receptors and a metabolic by misincorporating into cellular proteins. We have used zebrafish, an increasingly used model for neurodegenerative diseases, to further identify signaling components involved in BMAA-induced toxicity. Zebrafish embryos were exposed to sub-lethal dosages of BMAA and a label-free proteomics analysis was conducted on larvae 4 days post fertilization. The exposed larvae showed no developmental abnormalities, but a reduced heart rate and increased expression of GSK3 isoforms. Search towards a reviewed database containing 2968 entries identified 480 proteins. Only 17 of these were regulated 2-fold or more in the exposed larvae. Seven of these proteins could be associated to glutamate receptor signaling and recycling. The remaining nine have all been linked to disturbance in protein homeostasis, reactive oxygen species (ROS) development or neuronal cell death. We also found that BMAA influenced the endocannabinoid system by up-regulation of fatty acid amide hydrolase (FAAH) and that FAAH inhibitor URB597 reduced the BMAA effect on heart rate and GSK3 expression.

No MeSH data available.


Related in: MedlinePlus