Effect of staurosporine in the morphology and viability of cerebellar astrocytes: role of reactive oxygen species and NADPH oxidase

Abstract

Cell death implies morphological changes that may contribute to the progression of this process. In astrocytes, the mechanisms involving the cytoskeletal changes during cell death are not well explored. Although NADPH oxidase (NOX) has been described as being a critical factor in the production of ROS, not much information is available about the participation of NOX-derived ROS in the cell death of astrocytes and their role in the alterations of the cytoskeleton during the death of astrocytes. In this study, we have evaluated the participation of ROS in the death of cultured cerebellar astrocytes using staurosporine (St) as death inductor. We found that astrocytes express NOX1, NOX2, and NOX4. Also, St induced an early ROS production and NOX activation that participate in the death of astrocytes. These findings suggest that ROS produced by St is generated through NOX1 and NOX4. Finally, we showed that the reorganization of tubulin and actin induced by St is ROS independent and that St did not change the level of expression of these cytoskeletal proteins. We conclude that ROS produced by a NOX is required for cell death in astrocytes, but not for the morphological alterations induced by St.

Figure 1

Staurosporine reduces cerebellar astrocytes viability. (a) Cerebellar astrocytes were treated with staurosporine 0.1 μM, 0.25 μM, and 0.5 μM for 24 h and the cell viability was measured by MTT transformation as detailed in Section 2. (b) Cerebellar astrocytes were treated with staurosporine 0.5 μM for 6, 12, and 24 h and the cell viability was measured by MTT transformation as detailed in Section 2. Data are presented as mean ± SEM of four independent experiments. ∗ is significantly different from control (P < 0.05).

Figure 2

Reactive oxygen species are involved in cerebellar astrocytes death induced by St. (a) The levels of ROS were measured during different times by the oxidation of dihydroethidium (1 μM) as detailed in Section 2. Data are presented as mean ± SEM of four independent experiments. All points are significantly different from control (0 h) (P < 0.001). (b) Representative micrographs of cerebellar astrocytes treated with St (0.5 μM) for 24 in the presence or absence of MnTMPyP (50 μM) or AEBSF (50 μM). Cells were marked with calcein (green) and propidium iodide (red). Scale bar represents 50 μm. (c) Cell viability was determined by the percentage of calcein-positive cells from the total number of cells (calcein-positive cells plus propidium iodide-positive cells). Data are presented as mean ± SEM of three independent experiments.

Figure 3

NOX subunits are expressed in cerebellar astrocytes. NOX subunits expression was determined by RT-PCR assays in cultured astrocytes as detailed in Section 2 for NOX1 (268 bp), NOX2 (558 bp), and NOX4 (408 bp). Three independent assays were performed.

Figure 4

Staurosporine induces NOX activity. (a) NOX activity was evaluated at 2 h as detailed in Section 2. Data are presented as mean ± SEM of five independent experiments. ∗ is significantly different from control (P < 0.05). (b) ROS levels were determined in cerebellar astrocytes treated with St (0.5 μM) for 2 h in the presence or absence of AEBSF (50 μM) by measuring the oxidation of dihydroethidium (1 μM) as detailed in Section 2. Data are presented as mean ± SEM of three independent experiments.

Figure 5

Cell death induced by St is not mediated by NOX2 and NOX3. Cerebellar astrocytes were obtained from wild type mice and NOX2 KO and NOX3 KO mice. Cells were treated with St (0.5 μM) for 24 h and the cell viability was estimated as MTT transformation as detailed in Section 2. Data are presented as mean ± SEM of five independent experiments. ∗ is significantly different from control (P < 0.05).

Figure 6

Morphological changes of astrocytes induced by St are evidenced by the rearrangement of cytoskeletal proteins. Astrocytes were treated with St (0.5 μM) for 12 hours and then were labelled with rhodamine-phalloidin or immunostained for tubulin as detailed in Section 2. Representative images of phase contrast, rhodamine-phalloidin, and tubulin are shown in control and St treated astrocytes. Scale bar represents 50 μm.

Figure 7

Effect of St and hydrogen peroxide on the morphology of cerebellar astrocytes. Time-lapse images of astrocytes pretreated for 2 h with Euk-134 (20 μM) in the presence or absence of hydrogen peroxide (200 μM) and St (0.5 μM) were taken from the same field. Scale bar represents 50 μM.

Figure 8

Cytoskeletal rearrangements induced by St are not mediated by ROS. Cerebellar astrocytes were treated with St (0.5 μM) for 2 h in the presence or absence of the antioxidants MnTMPyP (50 μM) and Euk-134 (20 μM) and the NOX inhibitors DPI (520 nm) and AEBSF (50 μM). Immunostaining against β-tubulin (green) and staining with rhodamine-phalloidin (red) were performed as mentioned in Section 2. Scale bar represents 50 μM.

Figure 9

St does not induce changes in the expression of cytoskeletal proteins. Actin and α-tubulin levels were determined by Western blot assays as detailed in Section 2. Cerebellar astrocytes were treated with St (0.5 μM) in the presence or absence of the antioxidants MnTMPyP (50 μM) and Euk-134 (20 μM) and the NOX inhibitors DPI (520 nm) and AEBSF (50 μM). Data are presented as mean ± SEM of three independent experiments. No statistical differences were found among the treatments.