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Neurogenic and neurotrophic effects of BDNF peptides in mouse hippocampal primary neuronal cell cultures.

Cardenas-Aguayo Mdel C, Kazim SF, Grundke-Iqbal I, Iqbal K - PLoS ONE (2013)

Bottom Line: Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H(2)O(2)-treated E18 hippocampal cells.Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner.Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, United States of America.

ABSTRACT
The level of brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is down regulated in Alzheimer's disease (AD), Parkinson's disease (PD), depression, stress, and anxiety; conversely the level of this neurotrophin is increased in autism spectrum disorders. Thus, modulating the level of BDNF can be a potential therapeutic approach for nervous system pathologies. In the present study, we designed five different tetra peptides (peptides B-1 to B-5) corresponding to different active regions of BDNF. These tetra peptides were found to be non-toxic, and they induced the expression of neuronal markers in mouse embryonic day 18 (E18) primary hippocampal neuronal cultures. Additionally, peptide B-5 induced the expression of BDNF and its receptor, TrkB, suggesting a positive feedback mechanism. The BDNF peptides induced only a moderate activation (phosphorylation at Tyr 706) of the TrkB receptor, which could be blocked by the Trk's inhibitor, K252a. Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H(2)O(2)-treated E18 hippocampal cells. Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner. Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated.

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Activation of the TrkB receptor by BDNF peptides B-5 and B-3 can be blocked by the TrkB inhibitor, K252a.(A) Cells were pretreated with or without K252a for 1 h and then exposed to Peptide B-5 or Peptide B-3 at 1 µM or 20 ng/ml BDNF for 5 min. Western blots of pTrkB (Tyr 706), total TrkB and GAPDH included as loading control. (B) Densitometric quantitation of the Western-blots for pTrkB normalized to TrkB. Data are shown as mean ± standard deviation, n = 3. *p<0.05,**p<0.01, ***p<0.001. One-way ANOVA/post-hoc test/Student’s t-test.
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pone-0053596-g006: Activation of the TrkB receptor by BDNF peptides B-5 and B-3 can be blocked by the TrkB inhibitor, K252a.(A) Cells were pretreated with or without K252a for 1 h and then exposed to Peptide B-5 or Peptide B-3 at 1 µM or 20 ng/ml BDNF for 5 min. Western blots of pTrkB (Tyr 706), total TrkB and GAPDH included as loading control. (B) Densitometric quantitation of the Western-blots for pTrkB normalized to TrkB. Data are shown as mean ± standard deviation, n = 3. *p<0.05,**p<0.01, ***p<0.001. One-way ANOVA/post-hoc test/Student’s t-test.

Mentions: To further confirm the activation of the TrkB receptor by the peptides, we pretreated mouse primary E18 hippocampal cells with the Trk family inhibitor K252a for 1 h, and then added the growth factor BDNF (20 ng/ml) or the peptides B-5 or B-3 (at 1 µM) for 5 min and compared to vehicle-treated cells used as a control (C). By Western blots, we found a clear inhibition of the phosphorylation of TrkB at Tyrosine 706 by K252a (Fig. 6A, B). The increase in the phosphorylation of the TrkB receptor was significant for the treatment with B-5 and B-3 and BDNF (B-5, 1 µM, ANOVA, p = 0.0341; B-3, 1 µM, ANOVA, p = 0.019; BDNF, 20 ng/ml, ANOVA, p = 0.0003), but when cells were pretreated with K252a, there was a dramatic reduction in the activation of this receptor. However, BDNF, 20 ng/ml still induced a significant activation of TrkB (ANOVA, p = 0.032). These results confirmed that the peptides activated the TrkB receptor, and that this activation can be blocked by the Trk inhibitor, K252a. Total TrkB is shown as a reference, but we did not see a significant change in its expression. Since the treatment with the peptides was for a very short time (5 min), changes in the level of TrkB expression were not expected. There was an apparent specificity of the peptides B-5 and B-3 for activating TrkB receptor since we tested its activity on NIH 3T3 cells stably expressing TrkC, and neither the peptides nor BDNF were able to activate this receptor, which is normally activated by its ligand NT-3 (Fig. S3).


Neurogenic and neurotrophic effects of BDNF peptides in mouse hippocampal primary neuronal cell cultures.

Cardenas-Aguayo Mdel C, Kazim SF, Grundke-Iqbal I, Iqbal K - PLoS ONE (2013)

Activation of the TrkB receptor by BDNF peptides B-5 and B-3 can be blocked by the TrkB inhibitor, K252a.(A) Cells were pretreated with or without K252a for 1 h and then exposed to Peptide B-5 or Peptide B-3 at 1 µM or 20 ng/ml BDNF for 5 min. Western blots of pTrkB (Tyr 706), total TrkB and GAPDH included as loading control. (B) Densitometric quantitation of the Western-blots for pTrkB normalized to TrkB. Data are shown as mean ± standard deviation, n = 3. *p<0.05,**p<0.01, ***p<0.001. One-way ANOVA/post-hoc test/Student’s t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539976&req=5

pone-0053596-g006: Activation of the TrkB receptor by BDNF peptides B-5 and B-3 can be blocked by the TrkB inhibitor, K252a.(A) Cells were pretreated with or without K252a for 1 h and then exposed to Peptide B-5 or Peptide B-3 at 1 µM or 20 ng/ml BDNF for 5 min. Western blots of pTrkB (Tyr 706), total TrkB and GAPDH included as loading control. (B) Densitometric quantitation of the Western-blots for pTrkB normalized to TrkB. Data are shown as mean ± standard deviation, n = 3. *p<0.05,**p<0.01, ***p<0.001. One-way ANOVA/post-hoc test/Student’s t-test.
Mentions: To further confirm the activation of the TrkB receptor by the peptides, we pretreated mouse primary E18 hippocampal cells with the Trk family inhibitor K252a for 1 h, and then added the growth factor BDNF (20 ng/ml) or the peptides B-5 or B-3 (at 1 µM) for 5 min and compared to vehicle-treated cells used as a control (C). By Western blots, we found a clear inhibition of the phosphorylation of TrkB at Tyrosine 706 by K252a (Fig. 6A, B). The increase in the phosphorylation of the TrkB receptor was significant for the treatment with B-5 and B-3 and BDNF (B-5, 1 µM, ANOVA, p = 0.0341; B-3, 1 µM, ANOVA, p = 0.019; BDNF, 20 ng/ml, ANOVA, p = 0.0003), but when cells were pretreated with K252a, there was a dramatic reduction in the activation of this receptor. However, BDNF, 20 ng/ml still induced a significant activation of TrkB (ANOVA, p = 0.032). These results confirmed that the peptides activated the TrkB receptor, and that this activation can be blocked by the Trk inhibitor, K252a. Total TrkB is shown as a reference, but we did not see a significant change in its expression. Since the treatment with the peptides was for a very short time (5 min), changes in the level of TrkB expression were not expected. There was an apparent specificity of the peptides B-5 and B-3 for activating TrkB receptor since we tested its activity on NIH 3T3 cells stably expressing TrkC, and neither the peptides nor BDNF were able to activate this receptor, which is normally activated by its ligand NT-3 (Fig. S3).

Bottom Line: Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H(2)O(2)-treated E18 hippocampal cells.Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner.Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, United States of America.

ABSTRACT
The level of brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is down regulated in Alzheimer's disease (AD), Parkinson's disease (PD), depression, stress, and anxiety; conversely the level of this neurotrophin is increased in autism spectrum disorders. Thus, modulating the level of BDNF can be a potential therapeutic approach for nervous system pathologies. In the present study, we designed five different tetra peptides (peptides B-1 to B-5) corresponding to different active regions of BDNF. These tetra peptides were found to be non-toxic, and they induced the expression of neuronal markers in mouse embryonic day 18 (E18) primary hippocampal neuronal cultures. Additionally, peptide B-5 induced the expression of BDNF and its receptor, TrkB, suggesting a positive feedback mechanism. The BDNF peptides induced only a moderate activation (phosphorylation at Tyr 706) of the TrkB receptor, which could be blocked by the Trk's inhibitor, K252a. Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H(2)O(2)-treated E18 hippocampal cells. Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner. Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated.

Show MeSH
Related in: MedlinePlus