Figure 5.
Figure 5. Astrocyte responses to neurotrophins induce neurite fragmentation and neuronal death and do not require p75NTR. (A) Kinetics of changes in neuronal morphology after exposure to ACM as monitored by live imaging. Stimuli (IL1 or BDNF) or vehicle was given to astrocytes for 4 h and then removed. Cells received fresh medium and were cultured for a further 24 h. Then the ACM was collected and added to cultured neurons. Neurons were incubated with supernatants from nontreated (sNT), IL1-treated (10 ng/ml; sIL1), or BDNF-treated astrocytes (5 ng/ml; sBDNF) or to direct recombinant BDNF (5 ng/ml; rBDNF). Neuronal network was calculated as color density in the image field, and changes over time were visualized as the percentage of time point 0. Five videos were analyzed per treatment at four time points. (B) Fluorescence images showing representative stainings for β-tubulin, DAPI, and TUNEL in neuronal cultures after 24-h exposure to ACM. Bars, 50 µm. (C) Quantification of β-tubulin MFI, number of cells with neurites, length of neurites, and percentage of apoptotic cells in neuronal cultures exposed to ACM. Changes in β-tubulin MFI were calculated as the percentage of MFI in control cultures exposed to ACM from nontreated (sNT) astrocytes. Data were obtained in at least three independent experiments. (D) The same parameters as in C in neuronal cultures treated with recombinant BDNF (rBDNF) or IL1 (rIL1). Data were obtained in three independent experiments. (E) Astrocytes were treated with 5 ng/ml BDNF, NT-3, NT-4, or 10 ng/ml IL1. Neuronal cultures were exposed to ACM, and β-tubulin MFI and the percentage of apoptotic cells were evaluated. Data were obtained in three independent experiments. (F) Cytofluorimetric analysis of p75NTR surface expression on human cultured astrocytes. (G) 25 ng/ml BDNF or vehicle was given to astrocytes for 4 h in the presence of p75NTR blocking antibody or isotype control and then removed. Cells received fresh medium and were cultured for a further 24 h. Then the ACM was collected and added to cultured neurons. Graphs show quantification of β-tubulin MFI and the percentage of apoptotic cells. Three independent experiments were performed. All data are represented as mean ± SEM. ns, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Astrocyte responses to neurotrophins induce neurite fragmentation and neuronal death and do not require p75NTR. (A) Kinetics of changes in neuronal morphology after exposure to ACM as monitored by live imaging. Stimuli (IL1 or BDNF) or vehicle was given to astrocytes for 4 h and then removed. Cells received fresh medium and were cultured for a further 24 h. Then the ACM was collected and added to cultured neurons. Neurons were incubated with supernatants from nontreated (sNT), IL1-treated (10 ng/ml; sIL1), or BDNF-treated astrocytes (5 ng/ml; sBDNF) or to direct recombinant BDNF (5 ng/ml; rBDNF). Neuronal network was calculated as color density in the image field, and changes over time were visualized as the percentage of time point 0. Five videos were analyzed per treatment at four time points. (B) Fluorescence images showing representative stainings for β-tubulin, DAPI, and TUNEL in neuronal cultures after 24-h exposure to ACM. Bars, 50 µm. (C) Quantification of β-tubulin MFI, number of cells with neurites, length of neurites, and percentage of apoptotic cells in neuronal cultures exposed to ACM. Changes in β-tubulin MFI were calculated as the percentage of MFI in control cultures exposed to ACM from nontreated (sNT) astrocytes. Data were obtained in at least three independent experiments. (D) The same parameters as in C in neuronal cultures treated with recombinant BDNF (rBDNF) or IL1 (rIL1). Data were obtained in three independent experiments. (E) Astrocytes were treated with 5 ng/ml BDNF, NT-3, NT-4, or 10 ng/ml IL1. Neuronal cultures were exposed to ACM, and β-tubulin MFI and the percentage of apoptotic cells were evaluated. Data were obtained in three independent experiments. (F) Cytofluorimetric analysis of p75NTR surface expression on human cultured astrocytes. (G) 25 ng/ml BDNF or vehicle was given to astrocytes for 4 h in the presence of p75NTR blocking antibody or isotype control and then removed. Cells received fresh medium and were cultured for a further 24 h. Then the ACM was collected and added to cultured neurons. Graphs show quantification of β-tubulin MFI and the percentage of apoptotic cells. Three independent experiments were performed. All data are represented as mean ± SEM. ns, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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