Journal: Molecular Psychiatry
Article Title: Multimodal beneficial effects of BNN27, a nerve growth factor synthetic mimetic, in the 5xFAD mouse model of Alzheimer’s disease
doi: 10.1038/s41380-024-02833-w
Figure Lengend Snippet: A Illustrative graph that shows the ‘acute’ 48h administration of BNN27 and proneurotrophin in the E17.5 mature hippocampal neuronal culture and the initiation of the immunocytochemistry (ICC) experiment. B Representative images of immunofluorescence with TUNEL (green), Tuj1 (red), Hoechst (blue) conditions in the presence of Aβ vs the complete medium (CTR) condition. C Quantification of TUNEL (+) / Tuj1 (+) cells under 48 h of Aβ40 (5 μM) administration to E17.5 hippocampal neurons in the presence and absence of BNN27 (1-way ANOVA, Bonferroni multiple comparison test). p75NTR - not Trk receptors - seem to be implicated in the established effect. D Illustrative graph that shows the ‘acute’ 24h administration of BNN27 and proneurotrophin in the E17.5 mature hippocampal neuronal culture and the initiation of the immunocytochemistry (ICC) experiment. E Immunofluorescence with Cleaved Caspase-3 (green), Hoechst (blue) and [ F ] Tuj1 (green) conditions in the presence or absence of Aβ [Aβ40, 5 μM)] and/or p75NTR inhibitor [anti-p75 Receptor antibody (MC-192) Abcam], proBDNF and BNN27. G Quantification of TUNEL (+) / Tuj1 (+) cells under a 24 h administration of Aβ, p75NTR inhibitor treated as indicated at a 12DIV hippocampal neuronal cell culture (1-way ANOVA, Bonferroni multiple comparison test). H Quantification of TUNEL (+) / Tuj1 (+) cells under a 24 h administration of Aβ, panTrk inhibitor (AZD-1332, Alomone) treated as indicated at a 12DIV hippocampal neuronal cell culture (1-way ANOVA, Sidak’s multiple comparison test). I , J Immunofluorescence with Cleaved Caspase-3 (green), Hoechst (blue) and Tuj1 (green) conditions in the presence or absence of Aβ [Aβ40, 5 μM)] and/or panTrk inhibitor, NGF and BNN27. BNN27 affects the reciprocal extensive exposure to the Aβ40 toxicity at the E17.5 mature hippocampal neurons K Illustrative graph that shows the exact time periods for drug administrations and the immunocytochemistry (ICC) induction [ L ] Representative images of Cleaved Caspase-3 (red), Tuj1 (green), Hoechst (blue) staining of hippocampal 12DIV neurons after Aβ induction in the presence or absence of NGF, BNN27. The complete medium (CTR) condition serves as positive control. The images were taken at ×32 magnification (Scale bar, 20 μΜ). M Quantification of Cleaved Caspase-3 (+) / Tuj1 (+) after chronic administration (6 days) of NGF, BNN27 in order to test their ‘long term’ effects in the primary neuronal cell culture, with simultaneous injection of Aβ40 (5 μM) (1-way ANOVA followed by Sidak’s multiple comparison test) Data are expressed as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, n = 3; Scale bar = 20 μm.
Article Snippet: For immunohistochemistry and immunocytochemistry, the antibodies used were mouse anti- Aβ (6E10 clone, Covance, 1:500); rabbit anti-Doublecortin antibody (DCX, Abcam, 1:200); mouse anti-NeuN clone A60 (Sigma-Aldrich, 1:100); mouse anti- BrdU (MoBU-1.
Techniques: Immunocytochemistry, Immunofluorescence, TUNEL Assay, Comparison, Cell Culture, Staining, Positive Control, Injection
![<t>A</t> Illustrative graph that shows the ‘acute’ 48h administration of BNN27 and proneurotrophin in the E17.5 mature hippocampal neuronal culture and the initiation of the immunocytochemistry (ICC) experiment. B Representative images of immunofluorescence with TUNEL (green), Tuj1 (red), Hoechst (blue) conditions in the presence of Aβ vs the complete medium (CTR) condition. C Quantification of TUNEL (+) / Tuj1 (+) cells under 48 h of Aβ40 (5 μM) administration to E17.5 hippocampal neurons in the presence and absence of BNN27 (1-way ANOVA, Bonferroni multiple comparison test). p75NTR - not Trk receptors - seem to be implicated in the established effect. D Illustrative graph that shows the ‘acute’ 24h administration of BNN27 and proneurotrophin in the E17.5 mature hippocampal neuronal culture and the initiation of the immunocytochemistry (ICC) experiment. E Immunofluorescence with Cleaved Caspase-3 (green), Hoechst (blue) and [ F ] Tuj1 (green) conditions in the presence or absence of Aβ [Aβ40, 5 μM)] and/or p75NTR inhibitor [anti-p75 Receptor antibody (MC-192) Abcam], proBDNF and BNN27. G Quantification of TUNEL (+) / Tuj1 (+) cells under a 24 h administration of Aβ, p75NTR inhibitor treated as indicated at a 12DIV hippocampal neuronal cell culture (1-way ANOVA, Bonferroni multiple comparison test). H Quantification of TUNEL (+) / Tuj1 (+) cells under a 24 h administration of Aβ, panTrk inhibitor (AZD-1332, Alomone) treated as indicated at a 12DIV hippocampal neuronal cell culture (1-way ANOVA, Sidak’s multiple comparison test). I , J Immunofluorescence with Cleaved Caspase-3 (green), Hoechst (blue) and Tuj1 (green) conditions in the presence or absence of Aβ [Aβ40, 5 μM)] and/or panTrk inhibitor, NGF and BNN27. BNN27 affects the reciprocal extensive exposure to the Aβ40 toxicity at the E17.5 mature hippocampal neurons K Illustrative graph that shows the exact time periods for drug administrations and the immunocytochemistry (ICC) induction [ L ] Representative images of Cleaved Caspase-3 (red), Tuj1 (green), Hoechst (blue) staining of hippocampal 12DIV neurons after Aβ induction in the presence or absence of NGF, BNN27. The complete medium (CTR) condition serves as positive control. The images were taken at ×32 magnification (Scale bar, 20 μΜ). M Quantification of Cleaved Caspase-3 (+) / Tuj1 (+) after chronic administration (6 days) of NGF, BNN27 in order to test their ‘long term’ effects in the primary neuronal cell culture, with simultaneous injection of Aβ40 (5 μM) (1-way ANOVA followed by Sidak’s multiple comparison test) Data are expressed as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, n = 3; Scale bar = 20 μm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2300/pmc12092300/pmc12092300__41380_2024_2833_Fig3_HTML.jpg)
![Immunostaining for BrdU and NeuN in the dentate gyrus of the hippocampus of placebo and BNN27 treated 5xFAD animals [ A , B ]. There was an overall effect of the treatment ( P Two-way ANOVA = 0.0007) and genotype ( P Two-way ANOVA = 0.0011) in the number of BrdU+/NeuN+ neurons. Moreover, in the 5xFAD animals BNN27 treatment significantly ( P Bonferroni posttests = 0.0032) increases the number of BrdU positive neurons in the hippocampus compared to 5xFAD placebo animals [ C ]. Immunostaining for DCX in the dentate gyrus of the hippocampus of placebo and BNN27 treated animals [ D ]. There was an overall effect of the treatment ( P Two-way ANOVA = 0.0097) and genotype ( P Two-way ANOVA =0.0007) in the number of DCX+ neurons [2-way ANOVA for treatment and genotype]. Moreover, in the 5xFAD animals BNN27 treatment significantly ( P Bonferroni posttests = 0.011) increases the number of newly born neurons hippocampus [ E ]. Graphs showing mean ± SΕΜ; n = 4 * P < 0.05, ** P < 0.01, *** P < 0.0001, **** P < 0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2300/pmc12092300/pmc12092300__41380_2024_2833_Fig4_HTML.jpg)
![There was no detectable difference on the percentage of the BrdU(+) cells given the presence of BNN27 in the P7 Aβ-introduced NSC culture. A BrdU incorporation (green) evaluation of the Nestin(+) P7 NSCs (red). B Quantification of BrdU(+)/Nestin(+) cells under 48h induction of Aβ at the the P7 NSC hippocampal culture, in the presence or absence of NGF and BNN27 treatment, respectively. The complete condition depicts the positive control [1-way ANOVA for treatment and genotype, Sidak's multiple comparisons Post Hoc tests]. C Staining with CellTox Green Cytotoxicity Assay. D Evaluation of the in vitro cytotoxicity of Aβ42 oligomer as well as the potential 48 h neuroprotective activity of BDNF neurotrophin and BNN27 in the P7 NSCs of hippocampus. The complete medium (CTR) condition serves as positive control. [1-way ANOVA for treatment and genotype, Sidak's multiple comparisons Post Hoc tests]. Both Trk receptors and p75NTR seem to be implicated in the observed result. E Immunofluorescence with CellTox (green) and Hoechst (blue) conditions that shows the 24h administration in the P7 hippocampal neural stem cell culture in the presence or absence of Aβ42 (5 μM) and/or panTrk inhibitor (AZD-1332, Alomone), NGF or BNN27. F Quantification of CellTox (+) / Hoechst (+) cells under a 24 h administration of Aβ and/or panTrk inhibitor, treated as indicated at a P7 hippocampal neural stem cell culture (1-way ANOVA, Tukey multiple comparison test). G Immunofluorescence with CellTox (green) and Hoechst (blue) conditions that shows the 24h administration in the P7 hippocampal neural stem cell culture in the presence or absence of Aβ42 (5 μM) and/or p75NTR inhibitor [anti-p75 Receptor antibody (MC-192) Abcam], proBDNF or BNN27. H Quantification of CellTox (+) / Hoechst (+) cells under a 24 h presence or absence of Aβ and/or p75NTR inhibitor, treated as indicated at a P7 hippocampal neural stem cell culture (1-way ANOVA, Tukey multiple comparison test). Data are expressed as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, n = 3; Scale bar = 20 μm).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2300/pmc12092300/pmc12092300__41380_2024_2833_Fig5_HTML.jpg)