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Fig. 2. Specific neuronal-PKCßI activation can rescue SGNs from apoptosis induced by TFD. (A) SGNs deprived of trophic factors were cultured for 24 hours in the presence or absence of increasing concentrations of PMA. The neurons were fixed and immunostained with anti-ßIII tubulin antibody (TUJ1), as described in Materials and Methods, and the remaining number of viable neurons as a percentage of neurons in the untreated control condition was determined. All TUJ1-positive neurons were included in these counts, regardless of neurite length. Each such determination was performed in triplicate and repeated in three independent experiments. The mean value is shown in the figure; error bars in this and all subsequent figures indicate standard deviation (n=9; *P<0.05; ***P<0.001). (B) SGN cultures were treated or not with 100 nM PMA and neuronal apoptosis was determined by coupling the TUNEL bioassay to TUJ1 immunostaining 15 hours after completion of the spiral ganglia dissection. One hundred percent TUNEL-positive neurons was defined as the number of neurons presenting a TUNEL stain per total number of neurons in control cultures (n=6; ***P<0.001). The inset shows a representative confocal micrograph of TUJ1 (red)/TUNEL (green) double-stained P5 SGNs in control condition (-PMA) in which three out of five neurons are dying (arrows). Bar, 20 µm. (C) SGNs were cultured for 24 hours in the presence of 100 nM PMA and increasing concentrations of GF109203X, a specific inhibitor of PKC with high affinity for conventional isoforms. Cells were fixed and neuron viability was quantified, as determined in A (n=10; **P<0.01; ***P<0.001). (D) SGNs were incubated for 24 hours with 100 nM PMA (as a positive control) or increasing concentrations of the PMA inactive analogue, 4
-PMA. Neuronal cell counts were obtained as described in A (n=6; ***P<0.001). (E) Schematic representation of the neuroprotection paradigm performed in F. (F) SGNs were cultured in the presence or absence of PMA or PDBu (each at 100 nM) in conditions presented in E. SGNs were deprived of trophic factors for 4 hours. The neurons were then subjected to a PMA or PDBu treatment for 30 minutes or 20 hours. For each medium change, wells were rinsed three times in definite control medium, i.e. without trophic factor, to ensure the total elimination of agents from wells. Neuron viability was measured as determined in A (n=6-12; *P<0.05). (G,H) Representative confocal micrographs of expression and cellular redistribution of PKC and PKCßI in SG cells before and after stimulation with a 30 minute PMA treatment. SG cells were cultured for 4 hours in a definite control medium. At this time, cultures were treated with 100 nM PMA or vehicle (control condition) for 30 minutes. Next, the cells were fixed and immunostained with TUJ1 and PKC or PKCßI antibodies. In H, PKCßI is uniformly located exclusively in the cytoplasm of neurons in untreated cultures, whereas a 30 minute treatment with PMA induces its redistribution to the cell membrane (arrowheads). Insets show two TUJ1-positive neurons at higher magnification with a cytoplasmic (control condition) or a membranous (with PMA) PKCßI staining. The experiments were performed in duplicate and repeated on at least three different occasions, with similar results. Bars, 50 µm.
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