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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

Volcano plot of BMAA-induced protein changes observed by label-free quantitation.The protein expression ratio of BMAA/Control (log2 scale) in label-free quantitation was plotted against the −log10 of the probability calculated by t-test. Proteins with missing values are replaced by random numbers drawn from the normal distribution, and are marked as x in the graph. The dashed lines represent the applied threshold values (p-value ≤ 0.05, Fold change ≥ 2).
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f3: Volcano plot of BMAA-induced protein changes observed by label-free quantitation.The protein expression ratio of BMAA/Control (log2 scale) in label-free quantitation was plotted against the −log10 of the probability calculated by t-test. Proteins with missing values are replaced by random numbers drawn from the normal distribution, and are marked as x in the graph. The dashed lines represent the applied threshold values (p-value ≤ 0.05, Fold change ≥ 2).

Mentions: Protein extracts from total larvae were analyzed using a label-free proteomics approach to identify proteins that were differentially regulated in BMAA-exposed larvae compared to controls. We searched both towards the reviewed Danio rerio database with 2968 entries and the combined UniProtKB (reviewed & unreviewed) database with 58693 entries. In total, 480 (reviewed) and 2833 (UniProtKB) proteins were identified and quantified in the label-free approach. 295 (reviewed) and 1960 (UniProtKB) proteins meet the criteria (see material and methods section) for further statistical analysis to compare BMAA-exposed to control-injected larvae (Supplementary Tables S1–4). An overview of the number of proteins and peptides identified in each technical replicate can be seen in Supplementary Table S5. To limit the possibilities of focusing on false positive protein identifications we choose to work further on the dataset obtained with the curated database. Volcano plot in Fig. 3 shows the log2-fold change and p-value of all proteins in BMAA-exposed larvae compared with controls identified using the curated database. Of these, 17 proteins were found to be regulated 2-fold or more (Fig. 3 and Table 1). This included four proteins that were either found only in BMAA-exposed or only in controls. Imported values reflecting the lower detection limit of the mass spectrometry analysis were imported for proteins that were detected only in one condition in order to reflect fold regulation in Fig. 3. A multi-scatter plot showing the high reproducibility of the technical repeats can be seen in Supplementary Figure 1.


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)

Volcano plot of BMAA-induced protein changes observed by label-free quantitation.The protein expression ratio of BMAA/Control (log2 scale) in label-free quantitation was plotted against the −log10 of the probability calculated by t-test. Proteins with missing values are replaced by random numbers drawn from the normal distribution, and are marked as x in the graph. The dashed lines represent the applied threshold values (p-value ≤ 0.05, Fold change ≥ 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Volcano plot of BMAA-induced protein changes observed by label-free quantitation.The protein expression ratio of BMAA/Control (log2 scale) in label-free quantitation was plotted against the −log10 of the probability calculated by t-test. Proteins with missing values are replaced by random numbers drawn from the normal distribution, and are marked as x in the graph. The dashed lines represent the applied threshold values (p-value ≤ 0.05, Fold change ≥ 2).
Mentions: Protein extracts from total larvae were analyzed using a label-free proteomics approach to identify proteins that were differentially regulated in BMAA-exposed larvae compared to controls. We searched both towards the reviewed Danio rerio database with 2968 entries and the combined UniProtKB (reviewed & unreviewed) database with 58693 entries. In total, 480 (reviewed) and 2833 (UniProtKB) proteins were identified and quantified in the label-free approach. 295 (reviewed) and 1960 (UniProtKB) proteins meet the criteria (see material and methods section) for further statistical analysis to compare BMAA-exposed to control-injected larvae (Supplementary Tables S1–4). An overview of the number of proteins and peptides identified in each technical replicate can be seen in Supplementary Table S5. To limit the possibilities of focusing on false positive protein identifications we choose to work further on the dataset obtained with the curated database. Volcano plot in Fig. 3 shows the log2-fold change and p-value of all proteins in BMAA-exposed larvae compared with controls identified using the curated database. Of these, 17 proteins were found to be regulated 2-fold or more (Fig. 3 and Table 1). This included four proteins that were either found only in BMAA-exposed or only in controls. Imported values reflecting the lower detection limit of the mass spectrometry analysis were imported for proteins that were detected only in one condition in order to reflect fold regulation in Fig. 3. A multi-scatter plot showing the high reproducibility of the technical repeats can be seen in Supplementary Figure 1.

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