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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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(A) BDNF peptides B-5, B-4 and B-3 induce the expression of BDNF.Western blot analyses of cells treated with peptides B-5, B-4 and B-3, or with BDNF, or vehicle for 5 days, showed an increase in BDNF expression in cells treated with the peptides. A sample of adult mouse brain was included as a control for the migration of the bands corresponding to pro-BDNF and BDNF. (B) Densitometric quantitation of the Western blots developed with anti-BDNF. Data was normalized to GAPDH as loading control and then to 5 days control vehicle treated cells, C 5d. (C) Peptides B-5 and B-3 activated TrkB receptor in primary E18 hippocampal cells. Western blots showing phosphorylation of TrkB at Tyr 706 on treatment with Peptide B-5 (1 µM), Peptide B-3 (1 µM) or BDNF (20 ng/ml, 0.79 nM) for 1 h as compared to control treated cells, C. Lower blots show the levels of TrkB receptor and the levels of GAPDH as a loading control. (D) Densitometric analysis of the Western-blots for pTrkB normalized to TrkB, and TrkB normalized to GAPDH. Control was taken as 100 percent in each case. (E) BDNF peptides (B-5 and B-3) increased the expression of TrkB. Increase in expression of TrkB in TrkB stably-expressing NIH-3T3 fibroblast cells, as a function of time. Cells were treated for 5, 15 or 60 min with B-5 (1 µM), B-3 (1 µM), BDNF (20 ng/ml), or vehicle only (Control, C). Western blots of total TrkB, and GAPDH included as a loading control. (F) Densitometric quantitation of the Western blots for TrkB normalized to GAPDH. 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. Dashed line in (D) denotes that B-5 induction of TrkB expression almost approaches significance (p = 0.057, one-way ANOVA).
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pone-0053596-g005: (A) BDNF peptides B-5, B-4 and B-3 induce the expression of BDNF.Western blot analyses of cells treated with peptides B-5, B-4 and B-3, or with BDNF, or vehicle for 5 days, showed an increase in BDNF expression in cells treated with the peptides. A sample of adult mouse brain was included as a control for the migration of the bands corresponding to pro-BDNF and BDNF. (B) Densitometric quantitation of the Western blots developed with anti-BDNF. Data was normalized to GAPDH as loading control and then to 5 days control vehicle treated cells, C 5d. (C) Peptides B-5 and B-3 activated TrkB receptor in primary E18 hippocampal cells. Western blots showing phosphorylation of TrkB at Tyr 706 on treatment with Peptide B-5 (1 µM), Peptide B-3 (1 µM) or BDNF (20 ng/ml, 0.79 nM) for 1 h as compared to control treated cells, C. Lower blots show the levels of TrkB receptor and the levels of GAPDH as a loading control. (D) Densitometric analysis of the Western-blots for pTrkB normalized to TrkB, and TrkB normalized to GAPDH. Control was taken as 100 percent in each case. (E) BDNF peptides (B-5 and B-3) increased the expression of TrkB. Increase in expression of TrkB in TrkB stably-expressing NIH-3T3 fibroblast cells, as a function of time. Cells were treated for 5, 15 or 60 min with B-5 (1 µM), B-3 (1 µM), BDNF (20 ng/ml), or vehicle only (Control, C). Western blots of total TrkB, and GAPDH included as a loading control. (F) Densitometric quantitation of the Western blots for TrkB normalized to GAPDH. 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. Dashed line in (D) denotes that B-5 induction of TrkB expression almost approaches significance (p = 0.057, one-way ANOVA).

Mentions: To investigate the molecular mechanism by which the BDNF peptides promoted neurogenic/neurotrophic activities, we treated the hippocampal primary cultured neurons with the peptides or BDNF for 5 days and compared with the control medium treated cells. Peptides B-5, B-4 and B-3 induced the expression of BDNF, probably potentiating its signaling (Fig. 5A, B). The strongest induction of the expression of BDNF was produced by Peptide B-5 at a concentration of 0.1 µM (ANOVA, p<0.001); at 1 µM this effect of Peptide B-5 was lost. Peptide B-4 at a concentration of 1 µM and peptide B-3 at both 1 µM and 0.1 µM induced a significant increase in the expression of BDNF (ANOVA, p = 0.027).


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)

(A) BDNF peptides B-5, B-4 and B-3 induce the expression of BDNF.Western blot analyses of cells treated with peptides B-5, B-4 and B-3, or with BDNF, or vehicle for 5 days, showed an increase in BDNF expression in cells treated with the peptides. A sample of adult mouse brain was included as a control for the migration of the bands corresponding to pro-BDNF and BDNF. (B) Densitometric quantitation of the Western blots developed with anti-BDNF. Data was normalized to GAPDH as loading control and then to 5 days control vehicle treated cells, C 5d. (C) Peptides B-5 and B-3 activated TrkB receptor in primary E18 hippocampal cells. Western blots showing phosphorylation of TrkB at Tyr 706 on treatment with Peptide B-5 (1 µM), Peptide B-3 (1 µM) or BDNF (20 ng/ml, 0.79 nM) for 1 h as compared to control treated cells, C. Lower blots show the levels of TrkB receptor and the levels of GAPDH as a loading control. (D) Densitometric analysis of the Western-blots for pTrkB normalized to TrkB, and TrkB normalized to GAPDH. Control was taken as 100 percent in each case. (E) BDNF peptides (B-5 and B-3) increased the expression of TrkB. Increase in expression of TrkB in TrkB stably-expressing NIH-3T3 fibroblast cells, as a function of time. Cells were treated for 5, 15 or 60 min with B-5 (1 µM), B-3 (1 µM), BDNF (20 ng/ml), or vehicle only (Control, C). Western blots of total TrkB, and GAPDH included as a loading control. (F) Densitometric quantitation of the Western blots for TrkB normalized to GAPDH. 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. Dashed line in (D) denotes that B-5 induction of TrkB expression almost approaches significance (p = 0.057, one-way ANOVA).
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Related In: Results  -  Collection

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pone-0053596-g005: (A) BDNF peptides B-5, B-4 and B-3 induce the expression of BDNF.Western blot analyses of cells treated with peptides B-5, B-4 and B-3, or with BDNF, or vehicle for 5 days, showed an increase in BDNF expression in cells treated with the peptides. A sample of adult mouse brain was included as a control for the migration of the bands corresponding to pro-BDNF and BDNF. (B) Densitometric quantitation of the Western blots developed with anti-BDNF. Data was normalized to GAPDH as loading control and then to 5 days control vehicle treated cells, C 5d. (C) Peptides B-5 and B-3 activated TrkB receptor in primary E18 hippocampal cells. Western blots showing phosphorylation of TrkB at Tyr 706 on treatment with Peptide B-5 (1 µM), Peptide B-3 (1 µM) or BDNF (20 ng/ml, 0.79 nM) for 1 h as compared to control treated cells, C. Lower blots show the levels of TrkB receptor and the levels of GAPDH as a loading control. (D) Densitometric analysis of the Western-blots for pTrkB normalized to TrkB, and TrkB normalized to GAPDH. Control was taken as 100 percent in each case. (E) BDNF peptides (B-5 and B-3) increased the expression of TrkB. Increase in expression of TrkB in TrkB stably-expressing NIH-3T3 fibroblast cells, as a function of time. Cells were treated for 5, 15 or 60 min with B-5 (1 µM), B-3 (1 µM), BDNF (20 ng/ml), or vehicle only (Control, C). Western blots of total TrkB, and GAPDH included as a loading control. (F) Densitometric quantitation of the Western blots for TrkB normalized to GAPDH. 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. Dashed line in (D) denotes that B-5 induction of TrkB expression almost approaches significance (p = 0.057, one-way ANOVA).
Mentions: To investigate the molecular mechanism by which the BDNF peptides promoted neurogenic/neurotrophic activities, we treated the hippocampal primary cultured neurons with the peptides or BDNF for 5 days and compared with the control medium treated cells. Peptides B-5, B-4 and B-3 induced the expression of BDNF, probably potentiating its signaling (Fig. 5A, B). The strongest induction of the expression of BDNF was produced by Peptide B-5 at a concentration of 0.1 µM (ANOVA, p<0.001); at 1 µM this effect of Peptide B-5 was lost. Peptide B-4 at a concentration of 1 µM and peptide B-3 at both 1 µM and 0.1 µM induced a significant increase in the expression of BDNF (ANOVA, p = 0.027).

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