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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

Verification of mass spectrometry results.Total protein extracts (40 μg per lane) from BMAA- and control-injected larvae were separated by SDS-PAGE and transferred to PVDF membranes for Western blotting with the indicated antibodies. Ponceau-S staining was used as loading control. Blots show control and BMAA-injected samples from two individual experiments.
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f4: Verification of mass spectrometry results.Total protein extracts (40 μg per lane) from BMAA- and control-injected larvae were separated by SDS-PAGE and transferred to PVDF membranes for Western blotting with the indicated antibodies. Ponceau-S staining was used as loading control. Blots show control and BMAA-injected samples from two individual experiments.

Mentions: The possibility to verify the mass spectrometry data (Table 1) by Western blotting is somewhat restricted due the limited availability of zebrafish recognizing antibodies. Candidates were therefore chosen based on the number of covering tryptic peptides showing similar regulation and homology to proteins with available targeting antibodies. Western blot towards using anti-ANP32e showed two bands that both were down-regulated in BMAA-exposed animals, thus verifying the mass spectrometry data. Zebrafish ANP32e is predicted to be 28 kDa and is most likely the high intensity band. ANP32a which is highly homologues to ANP32e has a slight higher Mw (Fig. 4). Also, the BMAA-induced down-regulation in GAPDH and the Sec61a up-regulation were verified by Western blotting (Fig. 4).


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)

Verification of mass spectrometry results.Total protein extracts (40 μg per lane) from BMAA- and control-injected larvae were separated by SDS-PAGE and transferred to PVDF membranes for Western blotting with the indicated antibodies. Ponceau-S staining was used as loading control. Blots show 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

f4: Verification of mass spectrometry results.Total protein extracts (40 μg per lane) from BMAA- and control-injected larvae were separated by SDS-PAGE and transferred to PVDF membranes for Western blotting with the indicated antibodies. Ponceau-S staining was used as loading control. Blots show control and BMAA-injected samples from two individual experiments.
Mentions: The possibility to verify the mass spectrometry data (Table 1) by Western blotting is somewhat restricted due the limited availability of zebrafish recognizing antibodies. Candidates were therefore chosen based on the number of covering tryptic peptides showing similar regulation and homology to proteins with available targeting antibodies. Western blot towards using anti-ANP32e showed two bands that both were down-regulated in BMAA-exposed animals, thus verifying the mass spectrometry data. Zebrafish ANP32e is predicted to be 28 kDa and is most likely the high intensity band. ANP32a which is highly homologues to ANP32e has a slight higher Mw (Fig. 4). Also, the BMAA-induced down-regulation in GAPDH and the Sec61a up-regulation were verified by Western blotting (Fig. 4).

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