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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

FAAH inhibitor URB597 reduces the effect of BMAA.(A)FAAH inhibitor URB597 reduces the effect of BMAA on the heart rate (beats/min). Embryos were injected with BMAA or BMAA+URB597 at the one-cell stage. Heart rate was monitored at 3 dpf. Middle line in box represents the median, lower box bound the first quartile, and upper box bound the third quartile. n = 30–40 from three individual experiments, p < 0.001. (B) GSK3α/β expression in total lysates from injected larvae at 4 dpf. Ponceau-S staining was used as loading control. (C) GSK3α abundance. (D) GSK3β abundance. Data in C and D are expressed fold change relative to Ponceau-S expression in which bicarbonate-injected controls were used as the reference. Data are expressed as means +/− SEM from three independent experiments.
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f5: FAAH inhibitor URB597 reduces the effect of BMAA.(A)FAAH inhibitor URB597 reduces the effect of BMAA on the heart rate (beats/min). Embryos were injected with BMAA or BMAA+URB597 at the one-cell stage. Heart rate was monitored at 3 dpf. Middle line in box represents the median, lower box bound the first quartile, and upper box bound the third quartile. n = 30–40 from three individual experiments, p < 0.001. (B) GSK3α/β expression in total lysates from injected larvae at 4 dpf. Ponceau-S staining was used as loading control. (C) GSK3α abundance. (D) GSK3β abundance. Data in C and D are expressed fold change relative to Ponceau-S expression in which bicarbonate-injected controls were used as the reference. Data are expressed as means +/− SEM from three independent experiments.

Mentions: Our label free proteomics analysis showed FAAH2b to be up-regulated in BMAA-exposed animals (Table 1). Knock-out of FAAH in a mice model of ALS has been shown to inhibit the progression of neurodegeneration. It was therefore tempting to test if the FAAH inhibitor URB597 could inhibit BMAA-induced reduction in heart rate (Fig. 1) and increased GSK3 expression (Fig. 2). Indeed, URB597 protected against BMAA-induced reduction in heart rate (Fig. 5A). Western blots also showed a tendency, although not significant, of URB597 to inhibit BMAA-induced GSK3α and β increase (Fig. 5B–D).


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)

FAAH inhibitor URB597 reduces the effect of BMAA.(A)FAAH inhibitor URB597 reduces the effect of BMAA on the heart rate (beats/min). Embryos were injected with BMAA or BMAA+URB597 at the one-cell stage. Heart rate was monitored at 3 dpf. Middle line in box represents the median, lower box bound the first quartile, and upper box bound the third quartile. n = 30–40 from three individual experiments, p < 0.001. (B) GSK3α/β expression in total lysates from injected larvae at 4 dpf. Ponceau-S staining was used as loading control. (C) GSK3α abundance. (D) GSK3β abundance. Data in C and D are expressed fold change relative to Ponceau-S expression in which bicarbonate-injected controls were used as the reference. Data are expressed as means +/− SEM from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4940735&req=5

f5: FAAH inhibitor URB597 reduces the effect of BMAA.(A)FAAH inhibitor URB597 reduces the effect of BMAA on the heart rate (beats/min). Embryos were injected with BMAA or BMAA+URB597 at the one-cell stage. Heart rate was monitored at 3 dpf. Middle line in box represents the median, lower box bound the first quartile, and upper box bound the third quartile. n = 30–40 from three individual experiments, p < 0.001. (B) GSK3α/β expression in total lysates from injected larvae at 4 dpf. Ponceau-S staining was used as loading control. (C) GSK3α abundance. (D) GSK3β abundance. Data in C and D are expressed fold change relative to Ponceau-S expression in which bicarbonate-injected controls were used as the reference. Data are expressed as means +/− SEM from three independent experiments.
Mentions: Our label free proteomics analysis showed FAAH2b to be up-regulated in BMAA-exposed animals (Table 1). Knock-out of FAAH in a mice model of ALS has been shown to inhibit the progression of neurodegeneration. It was therefore tempting to test if the FAAH inhibitor URB597 could inhibit BMAA-induced reduction in heart rate (Fig. 1) and increased GSK3 expression (Fig. 2). Indeed, URB597 protected against BMAA-induced reduction in heart rate (Fig. 5A). Western blots also showed a tendency, although not significant, of URB597 to inhibit BMAA-induced GSK3α and β increase (Fig. 5B–D).

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