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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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BDNF peptides B-5, B-4, and B-3 are neurogenic and neurotrophic.(A–C) Peptide B-5 induces the expression of the neuronal markers β-III-tubulin, MAP2, NFM, and NeuN in E18 primary hippocampal cell cultures. Representative confocal images illustrating double immunolabeling of β–III-tubulin (green) and MAP2 (red) and NFM (green) and NeuN (red) in cells treated for 5 days with (A) Peptide B-5 at 1 µM, (B) with BDNF, 20 ng/ml (0.79 nM) for 5 days, and,(C) vehicle only (control, C). TOPRO-3 (blue) was used to stain the nuclei. Magnification bar = 10 µm. The bottom panel shows the level of fluorescence intensity using Image J software to measure the level of expression. (D, E) BDNF peptides (B-5, B-4, and B-3) induce the expression of neuronal markers in E18 primary hippocampal neurons. (D) Representative Western blots showing an increase in the expression of the neuronal markers PSD95, NeuN, NFM, and MAP-2 in cells treated with the peptides (B5, B-4 and B-3) at concentrations of 0.1 µM and 1 µM or BDNF at concentrations of 20 or 100 ng/ml (0.79 or 3.95 nM respectively). GPADH was used as a loading control. (E) Quantification of the Western blots of neuronal markers shown in D. The integrated density value of the bands in Western blots was determined using densitometry (Fuji software, Multi Gauge, Version 3.0), and data was normalized to GAPDH and to control (medium treated cells for 5 days, C 5d). Data are shown as mean ± standard deviation, n = 3. 10% SDS-PAGE gels. *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-g003: BDNF peptides B-5, B-4, and B-3 are neurogenic and neurotrophic.(A–C) Peptide B-5 induces the expression of the neuronal markers β-III-tubulin, MAP2, NFM, and NeuN in E18 primary hippocampal cell cultures. Representative confocal images illustrating double immunolabeling of β–III-tubulin (green) and MAP2 (red) and NFM (green) and NeuN (red) in cells treated for 5 days with (A) Peptide B-5 at 1 µM, (B) with BDNF, 20 ng/ml (0.79 nM) for 5 days, and,(C) vehicle only (control, C). TOPRO-3 (blue) was used to stain the nuclei. Magnification bar = 10 µm. The bottom panel shows the level of fluorescence intensity using Image J software to measure the level of expression. (D, E) BDNF peptides (B-5, B-4, and B-3) induce the expression of neuronal markers in E18 primary hippocampal neurons. (D) Representative Western blots showing an increase in the expression of the neuronal markers PSD95, NeuN, NFM, and MAP-2 in cells treated with the peptides (B5, B-4 and B-3) at concentrations of 0.1 µM and 1 µM or BDNF at concentrations of 20 or 100 ng/ml (0.79 or 3.95 nM respectively). GPADH was used as a loading control. (E) Quantification of the Western blots of neuronal markers shown in D. The integrated density value of the bands in Western blots was determined using densitometry (Fuji software, Multi Gauge, Version 3.0), and data was normalized to GAPDH and to control (medium treated cells for 5 days, C 5d). Data are shown as mean ± standard deviation, n = 3. 10% SDS-PAGE gels. *p<0.05, **p<0.01, ***p<0.001. One-way ANOVA/post-hoc test/Student’s t test.

Mentions: Immunocytochemical studies revealed that peptide B-5 at a concentration of 1 µM induced an increase in the level of expression of MAP2, β-III-tubulin, NFM, and NeuN in mouse E18 hippocampal neurons after 5 days of treatment (Fig. 3A), as compared to vehicle-treated cells (Fig. 3C). The increase in the fluorescence intensity level of MAP2, NFM and NeuN staining with peptide B-5 was similar to the one obtained when cells were treated with BDNF, 20 ng/ml, (0.79 nM) (Fig. 3B), suggesting that the peptides could mimic at least in part the effects of the parent growth factor (BDNF). The fact that the cells treated with Peptide B-5 were NeuN-positive could mean that these cells were terminally differentiated and for that reason they could also be functional, since they were also positive for other neuronal markers, such as β–III-tubulin and MAP2. Peptide B-3 had similar effects on the expression of the neuronal markers (data not shown). To further elaborate the increase in the level of expression of the neuronal markers, we obtained the rainbow scale images corresponding to the confocal images in Figure 3 (Fig. S2). In the rainbow images, the warm colors (like red and yellow) represent the higher level of expression of the neuronal marker analyzed, and the cold colors (like blue) represent the lowest level of expression of the marker. White represents the highest level of expression whereas black represents no expression at all.


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)

BDNF peptides B-5, B-4, and B-3 are neurogenic and neurotrophic.(A–C) Peptide B-5 induces the expression of the neuronal markers β-III-tubulin, MAP2, NFM, and NeuN in E18 primary hippocampal cell cultures. Representative confocal images illustrating double immunolabeling of β–III-tubulin (green) and MAP2 (red) and NFM (green) and NeuN (red) in cells treated for 5 days with (A) Peptide B-5 at 1 µM, (B) with BDNF, 20 ng/ml (0.79 nM) for 5 days, and,(C) vehicle only (control, C). TOPRO-3 (blue) was used to stain the nuclei. Magnification bar = 10 µm. The bottom panel shows the level of fluorescence intensity using Image J software to measure the level of expression. (D, E) BDNF peptides (B-5, B-4, and B-3) induce the expression of neuronal markers in E18 primary hippocampal neurons. (D) Representative Western blots showing an increase in the expression of the neuronal markers PSD95, NeuN, NFM, and MAP-2 in cells treated with the peptides (B5, B-4 and B-3) at concentrations of 0.1 µM and 1 µM or BDNF at concentrations of 20 or 100 ng/ml (0.79 or 3.95 nM respectively). GPADH was used as a loading control. (E) Quantification of the Western blots of neuronal markers shown in D. The integrated density value of the bands in Western blots was determined using densitometry (Fuji software, Multi Gauge, Version 3.0), and data was normalized to GAPDH and to control (medium treated cells for 5 days, C 5d). Data are shown as mean ± standard deviation, n = 3. 10% SDS-PAGE gels. *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-g003: BDNF peptides B-5, B-4, and B-3 are neurogenic and neurotrophic.(A–C) Peptide B-5 induces the expression of the neuronal markers β-III-tubulin, MAP2, NFM, and NeuN in E18 primary hippocampal cell cultures. Representative confocal images illustrating double immunolabeling of β–III-tubulin (green) and MAP2 (red) and NFM (green) and NeuN (red) in cells treated for 5 days with (A) Peptide B-5 at 1 µM, (B) with BDNF, 20 ng/ml (0.79 nM) for 5 days, and,(C) vehicle only (control, C). TOPRO-3 (blue) was used to stain the nuclei. Magnification bar = 10 µm. The bottom panel shows the level of fluorescence intensity using Image J software to measure the level of expression. (D, E) BDNF peptides (B-5, B-4, and B-3) induce the expression of neuronal markers in E18 primary hippocampal neurons. (D) Representative Western blots showing an increase in the expression of the neuronal markers PSD95, NeuN, NFM, and MAP-2 in cells treated with the peptides (B5, B-4 and B-3) at concentrations of 0.1 µM and 1 µM or BDNF at concentrations of 20 or 100 ng/ml (0.79 or 3.95 nM respectively). GPADH was used as a loading control. (E) Quantification of the Western blots of neuronal markers shown in D. The integrated density value of the bands in Western blots was determined using densitometry (Fuji software, Multi Gauge, Version 3.0), and data was normalized to GAPDH and to control (medium treated cells for 5 days, C 5d). Data are shown as mean ± standard deviation, n = 3. 10% SDS-PAGE gels. *p<0.05, **p<0.01, ***p<0.001. One-way ANOVA/post-hoc test/Student’s t test.
Mentions: Immunocytochemical studies revealed that peptide B-5 at a concentration of 1 µM induced an increase in the level of expression of MAP2, β-III-tubulin, NFM, and NeuN in mouse E18 hippocampal neurons after 5 days of treatment (Fig. 3A), as compared to vehicle-treated cells (Fig. 3C). The increase in the fluorescence intensity level of MAP2, NFM and NeuN staining with peptide B-5 was similar to the one obtained when cells were treated with BDNF, 20 ng/ml, (0.79 nM) (Fig. 3B), suggesting that the peptides could mimic at least in part the effects of the parent growth factor (BDNF). The fact that the cells treated with Peptide B-5 were NeuN-positive could mean that these cells were terminally differentiated and for that reason they could also be functional, since they were also positive for other neuronal markers, such as β–III-tubulin and MAP2. Peptide B-3 had similar effects on the expression of the neuronal markers (data not shown). To further elaborate the increase in the level of expression of the neuronal markers, we obtained the rainbow scale images corresponding to the confocal images in Figure 3 (Fig. S2). In the rainbow images, the warm colors (like red and yellow) represent the higher level of expression of the neuronal marker analyzed, and the cold colors (like blue) represent the lowest level of expression of the marker. White represents the highest level of expression whereas black represents no expression at all.

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